Compounds and their methods of use

ABSTRACT

Compounds and compositions comprising compounds that inhibit glutaminase are described herein. Also described herein are methods of using the compounds that inhibit glutaminase in the treatment of cancer.

CLAIM OF PRIORITY

This application claims priority from International Patent ApplicationNumber PCT/CN2012/085023, filed Nov. 22, 2012 and International PatentApplication Number PCT/CN2013/000294, filed Mar. 15, 2013, each of whichis incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

Cancer cells rely primarily on glycolysis to generate cellular energyand biochemical intermediates for biosynthesis of lipids andnucleotides, while the majority of “normal” cells in adult tissuesutilize aerobic respiration. This fundamental difference in cellularmetabolism between cancer cells and normal cells is termed the WarburgEffect. As a result of this difference, pyruvate generated via theglycolytic pathway is converted to lactic acid, rather than being usedto acetyl-CoA and eventually, the citrate utilized in a normal citricacid cycle. To compensate for these energetic changes and to maintain acitric acid cycle, cancer cells rely on glutamine metabolism which isachieved through an elevation of glutaminase activity. Exploitation ofthis phenomenon can be achieved by inhibition of this elevatedglutaminase activity.

SUMMARY OF INVENTION

Described herein are heterocyclic containing, pharmaceuticallyacceptable salts, solvates, and hydrates thereof. The compounds can beused to treat a disorder described herein, for example, by inhibitingglutaminase in a patient. Also provided are compositions (e.g.,pharmaceutical compositions) comprising a compound provided herewith andthe use of such compositions in methods of treating diseases andconditions, for example, that are associated with the aberrant functionof glutaminase or elevated activity of glutaminase, including, e.g.,cancer.

In one embodiment, provided is a compound of formula (I) or apharmaceutically acceptable salt thereof:

wherein

X is C₃-C₇ cycloalkylene;

each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, providedthat at least one of W, Y and Z is not —CH═;

each R¹ and R² is independently —NH₂, —N(R³)—C(O)—R⁴, —C(O)—N(R³)—R⁴,—N(R³)—C(O)—O—R⁴, —N(R³)—C(O)—N(R³)—R⁴ or —N(R³)—C(O)—SR⁴;

each R³ is independently hydrogen, C₁₋₆ alkyl or aryl;

each R⁴ is independently C₁₋₆ alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, orheterocyclyl, each of which is substituted with 0-3 occurrences of R⁵;

each R⁵ is independently C₁₋₆ alkyl, C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆alkoxy, C₁₋₆ thioalkoxy, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇cycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclylalkyl, heterocyclyl, cyano, halo, oxo, —OH, —OCF₃, —OCHF₂,—SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl, —C(O)N(R⁷)₂,—N(R⁷)S(O)₁₋₂—C₁₋₆ alkyl, —S(O)₂N(R⁷)₂, —N(R⁷)₂, —C₁₋₆ alkylene-N(R⁷)₂,wherein said alkyl, C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆ alkoxy, C₁₋₆thioalkoxy, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkylalkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl,—SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl, —C(O)N(R⁷)₂,—N(R⁷)S(O)₁₋₂—C₁₋₆ alkyl, —S(O)₂N(R⁷)₂, —N(R⁷)₂, or —C₁₋₆alkylene-N(R⁷)₂ is optionally substituted with 0-3 occurrences of R⁸; ortwo adjacent R⁵ moieties, taken together with the atoms to which theyare attached form a cycloalkyl or heterocyclyl;

each R⁶ is independently hydrogen, fluoro, C₁₋₆ alkyl, —OH, —NH₂,—NH(CH₃), —N(CH₃)₂, or C₁₋₆ alkoxy;

each R⁷ is independently hydrogen or C₁₋₆ alkyl;

each R⁸ is independently halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OH, —N(R⁷)₂,or C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆ alkoxy, CN, NO₂, —N(R⁷)—C(O)—C₁₋₆alkyl, —C(O)N(R⁷)₂, —N(R⁷)S(O)₁₋₂C₁₋₆ alkyl, or —S(O)₂N(R⁷)₂;

m is 0, 1, or 2;

n is 0, 1, or 2;

o is 1, 2 or 3; and

p is 1, 2 or 3; provided that (1) when X is unsubstituted cyclopropyl,R¹ and R² are not both NH-phenyl; and (2) X is other than substitutedcyclobutyl or substituted cyclopentyl.

In another embodiment, provided is a composition comprising a compoundof formula (I) or a pharmaceutically acceptable salt thereof. In someembodiments, the composition is a pharmaceutical composition.

In another embodiment, provided herein is a method for treating orpreventing a disease, condition or disorder as described (e.g.,treating) herein comprising administering a compound described herein, apharmaceutically acceptable salt thereof or a pharmaceutical compositioncomprising a compound described herein or a pharmaceutically acceptablesalt thereof.

In another embodiment, provided herein is a method of inhibitingglutaminase, e.g, in a patient in need thereof. In some embodiments,provided here is reducing the level of the product of glutaminase in asubject, e.g., a patient in need thereof. The methods includeadministering an effective amount of a compound described herein or apharmaceutically acceptable salt thereof to a subject in need thereof,thereby inhibiting the level of glutaminase in the subject.

In another embodiment, provided herein is a method of treating a subjectsuffering from or susceptible to a disease or disorder associated withthe aberrant function of glutaminase or elevated activity of glutaminasein a subject in need thereof. The method comprises the step ofadministering an effective amount of a compound described herein to thesubject in need thereof, thereby treating, preventing or amelioratingthe disease or disorder in the subject. In certain embodiments, thecompound is provided in a pharmaceutical composition. In certainembodiments, the method includes identifying or selecting a subject whowould benefit from inhibiting glutaminase or decreasing the level ofglutaminase. E.g., the subject can be identified on the basis of thelevel of glutaminase activity in a cell or tissue sample of the subjectfor treatment of cancer associated with aberrant glutaminase function oractivity. In another embodiment, the selected subject is a patientsuffering from or susceptible to a disorder or disease identifiedherein, e.g., a disorder characterized by unwanted cell growth orproliferation, e.g., cancer or other neoplastic disorders.

In another embodiment, provided herein is a method for treating cancerin a subject, the method comprising: optionally, acquiring a subjectsample; acquiring an evaluation of or evaluating the subject sample,wherein the subject sample is characterized by i) a low level ofE-cadherin expression compared to a reference standard, ii) a high levelof vimentin expression compared to a reference standard, or iii) a lowor decreased level of pyruvate carboxylase expression; and administeringto the subject in need thereof a therapeutically effective amount of acompound described here. In some embodiments, the subject sample ischaracterized by i) a low level of E-cadherin expression compared to areference standard and ii) a high level of vimentin expression comparedto a reference standard. In some embodiments, the subject sample ischaracterized or further characterized by low or decreased levels ofpyruvate carboxylase expression compared to a reference standard.

In another embodiment, provided herein is a method for treating cancerin a subject characterized by i) a low level of E-cadherin expressioncompared to a reference standard, ii) a high level of vimentinexpression compared to a reference standard, or iii) a low or decreasedlevel of pyruvate carboxylase expression; comprising administering tothe subject in need thereof a therapeutically effective amount of acompound described here. In some embodiments, the subject ischaracterized by i) a low level of E-cadherin expression compared to areference standard and ii) a high level of vimentin expression comparedto a reference standard. In some embodiments, the subject ischaracterized or further characterized by low or decreased levels ofpyruvate carboxylase expression compared to a reference standard.

DETAILED DESCRIPTION

The details of construction and the arrangement of components set forthin the following description or illustrated in the drawings are notmeant to be limiting. Embodiments can be practiced or carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing”,“involving”, and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Compounds

Described herein are compounds and compositions that inhibitglutaminase. Compounds that inhibit glutaminase, can be used to treatdisorders such as neoplastic disorders (e.g., cancer).

In one embodiment, provided is a compound of formula (I) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof:

wherein

X is C₃-C₇ cycloalkylene;

each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, providedthat at least one of W, Y and Z is not —CH═;

each R¹ and R² is independently —NH₂, —N(R³)—C(O)—R⁴, —C(O)—N(R³)—R⁴,—N(R³)—C(O)—O—R⁴, —N(R³)—C(O)—N(R³)—R⁴ or —N(R³)—C(O)—SR⁴;

each R³ is independently hydrogen, C₁₋₆ alkyl or aryl;

each R⁴ is independently C₁₋₆ alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, orheterocyclyl, each of which is substituted with 0-3 occurrences of R⁵;

each R⁵ is independently C₁₋₆ alkyl, C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆alkoxy, C₁₋₆ thioalkoxy, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇cycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclylalkyl, heterocyclyl, cyano, halo, oxo, —OH, —OCF₃, —OCHF₂,—SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl, —C(O)N(R⁷)₂,—N(R⁷)S(O)₁₋₂—C₁₋₆ alkyl, —S(O)₂N(R⁷)₂, —N(R⁷)₂, —C₁₋₆ alkylene-N(R⁷)₂,wherein said alkyl, C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆ alkoxy, C₁₋₆thioalkoxy, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkylalkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl,—SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl, —C(O)N(R⁷)₂,—N(R⁷)S(O)₁₋₂—C₁₋₆ alkyl, —S(O)₂N(R⁷)₂, —N(R⁷)₂, or —C₁₋₆alkylene-N(R⁷)₂ is optionally substituted with 0-3 occurrences of R⁸; ortwo adjacent R⁵ moieties, taken together with the atoms to which theyare attached form a cycloalkyl or heterocyclyl;

each R⁶ is independently hydrogen, fluoro, C₁₋₆ alkyl, —OH, —NH₂,—NH(CH₃), —N(CH₃)₂, or C₁₋₆ alkoxy;

each R⁷ is independently hydrogen or C₁₋₆ alkyl;

each R⁸ is independently halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OH, —N(R⁷)₂,or C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆ alkoxy, CN, NO₂, —N(R⁷)—C(O)—C₁₋₆alkyl, —C(O)N(R⁷)₂, —N(R⁷)S(O)₁₋₂C₁₋₆ alkyl, or —S(O)₂N(R⁷)₂;

m is 0, 1, or 2;

n is 0, 1, or 2;

o is 1, 2 or 3; and

p is 1, 2 or 3; provided that (1) when X is unsubstituted cyclopropyl,R¹ and R² are not both NH-phenyl; and (2) X is other than substitutedcyclobutyl or substituted cyclopentyl.

In one embodiment, provided is a compound of formula (I) or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof:

wherein

X is C₃-C₇ cycloalkylene;

each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, providedthat at least one of W, Y and Z is not —CH═;

each R¹ and R² is independently —NH₂, —N(R³)—C(O)—R⁴, —C(O)—N(R³)—R⁴,—N(R³)—C(O)—O—R⁴, —N(R³)—C(O)—N(R³)—R⁴ or —N(R³)—C(O)—SR⁴;

each R³ is independently hydrogen, C₁₋₆ alkyl or aryl;

each R⁴ is independently C₁₋₆ alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, orheterocyclyl, each of which is substituted with 0-3 occurrences of R⁵;

each R⁵ is independently C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ thioalkoxy, C₁₋₆haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkylalkyl, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cyano, halo,oxo, —OH, —OCF₃, —OCHF₂, —SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl,—N(R⁷)₂, or two adjacent R⁵ moieties, taken together with the atoms towhich they are attached form a heterocyclyl;

each R⁶ is independently hydrogen, fluoro, C₁₋₆ alkyl, —OH, —NH₂,—NH(CH₃), —N(CH₃)₂, or C₁₋₆ alkoxy;

each R⁷ is independently hydrogen or C₁₋₆ alkyl;

m is 0, 1, or 2;

n is 0, 1, or 2;

o is 1, 2 or 3; and

p is 1, 2 or 3; provided that (1) when X is unsubstituted cyclopropyl,R¹ and R² are not both NH-phenyl; and (2) X is other than substitutedcyclobutyl or substituted cyclopentyl.

In some embodiments, X is unsubstituted cyclopropyl. In someembodiments, X is unsubstituted cyclobutyl. In some embodiments, X isunsubstituted cyclopentyl. In some embodiments, X is cyclohexyl. In someembodiments, X is cycloheptyl.

In some embodiments, each Y is —N═. In some embodiments, each Z is —N═.In some embodiments, each W is —S—. In some aspects of theseembodiments, each W is —S—, each Y is —N═ and each Z is —N═.

In some embodiments, o is 1. In some embodiments, p is 1. In someembodiments, o is 1 and p is 1.

In some embodiments, m is 0. In some embodiments, n is 0. In someembodiments, m is 0 and n is 0. In some embodiments, R¹ and R² are thesame. In some embodiments, R¹ and R² are different.

In some embodiments, m is 1. In some embodiments, n is 1. In someembodiments, n is 1 and m is 1. In some aspects of these embodiments,each R⁶ is hydrogen. In some embodiments, R¹ and R² are the same. Insome embodiments, R¹ and R² are different.

In some embodiments, R¹ and R² are each —N(R³)—C(O)—R⁴ wherein each R³is hydrogen and each R⁴ is aralkyl or heteroaralkyl, each of which issubstituted with 0-3 occurrences of R⁵. In some aspects of theseembodiments, R¹ and R² are the same.

In some embodiments, R¹ and R² are each —N(R³)—C(O)—R⁴ wherein each R³is hydrogen. In some aspects of these embodiments, each R⁴ is aralkylsubstituted with 0-3 occurrences of R⁵. In some aspects of theseembodiments, R¹ and R² are the same.

In some aspects of these embodiments, each R⁴ is aralkyl (e.g., benzyl)substituted with 0 occurrences of R⁵. In some aspects of theseembodiments, each R⁴ is aralkyl (e.g., benzyl) substituted with oneoccurrence of R⁵. In some further aspects of these embodiments, each R⁵is —N(CH₃)₂. In other further aspects of these embodiments, each R⁵ isC₁₋₆ alkoxy (e.g., methoxy or isopropoxy). In other further aspects ofthese embodiments, each R⁵ is halo (e.g., fluoro or chloro). In otherfurther aspects of these embodiments, each R⁵ is —NH₂. In other furtheraspects of these embodiments, each R⁵ is —SO₂—CH₃. In other furtheraspects of these embodiments, each R⁵ is —NHC(O)CH₃. In other furtheraspects of these embodiments, each R⁵ is —NO₂. In other further aspectsof these embodiments, each R⁵ is cyano. In other further aspects ofthese embodiments, each R⁵ is C₁₋₆ haloalkoxy (e.g., trifluoromethoxy).In other further aspects of these embodiments, each R⁵ is C₁₋₆ haloalkyl(e.g., trifluoromethyl). In other further aspects of these embodiments,each R⁵ is C₁₋₆ alkyl (e.g., methyl). In some aspects of theseembodiments, each R⁴ is aralkyl (e.g., benzyl) substituted with twooccurrences of R⁵. In some further aspects of these embodiments, two R⁵are halo (e.g., fluoro) and the other two R⁵ are C₁₋₆ alkoxy (e.g.,methoxy). In other further aspects of these embodiments, each R⁵ is halo(e.g., fluoro). In other further aspects of these embodiments, each R⁵is C₁₋₆ alkoxy (e.g., methoxy). In other further aspects, of theseembodiments, two adjacent R⁵ moieties are taken together with the atomsto which they are attached to form a heterocyclyl ring resulting in amoiety of the following structure:

In some aspects of these embodiments, each R⁴ is heteroaralkyl (e.g.,2-pyridinylmethyl, 2-pyridinylethyl, 3-pyridinylmethyl,4-pyridinylmethyl, 2-pyrazinylmethyl, 2-thiophenylmethyl,2-indolylmethyl, 4-indolylmethyl, 2-pyrimidinylmethyl or2-thiazolylmethyl) substituted with 0-3 occurrences of R⁵. In someaspects of these embodiments, each R⁴ is heteroaralkyl (e.g.,2-pyridinylmethyl, 2-pyridinylethyl, 3-pyridinylmethyl,4-pyridinylmethyl, 2-pyrazinylmethyl, 2-thiophenylmethyl,2-indolylmethyl, 3-indolylmethyl, 4-indolylmethyl, 2-pyrimidinylmethylor 2-thiazolylmethyl) substituted with 0 occurrences of R⁵. In otheraspects of these embodiments, each R⁴ is heteroaralkyl (e.g.,5-isoxazolyl, 2-pyridinylmethyl or 3-indolylmethyl) substituted with oneoccurrence of R⁵. In some further aspects of these embodiments, each R⁵is C₁₋₆ alkyl (e.g., methyl). In other further aspects of theseembodiments, each R⁵ is C₁₋₆ alkoxy (e.g., methoxy). In other furtheraspects of these embodiments, each R⁵ is cyano. In other further aspectsof these embodiments, each R⁵ is —N(CH₃)₂. In other further aspects ofthese embodiments, each R⁵ is —NHC(O)CH₃. In other further aspects ofthese embodiments, each R⁵ is halo (e.g., bromo).

In some aspects of these embodiments, each R⁴ is C₁₋₆ alkyl (e.g.,methyl, ethyl, n-propyl or isopropyl) substituted with 0-3 occurrencesof R⁵. In some aspects of these embodiments, each R⁴ is C₁₋₆ alkyl(e.g., methyl, ethyl, n-propyl or isopropyl) substituted with 0occurrences of R⁵. In other aspects of these embodiments, each R⁴ isC₁₋₆ alkyl (e.g., methyl, ethyl or tert-butyl) substituted with oneoccurrence of R⁵. In some further aspects of these embodiments, each R⁵is C₁₋₆ thioalkoxy (e.g., thiomethoxy). In other further aspects ofthese embodiments, each R⁵ is C₁₋₆ haloalkyl (e.g., trifluoromethyl). Inother further aspects of these embodiments, each R⁵ is —OH.

In some aspects of these embodiments, each R⁴ is aryl (e.g., phenyl)substituted with 0-3 occurrences of R⁵. In some aspects of theseembodiments, each R⁴ is aryl (e.g., phenyl) substituted with 0occurrences of R⁵.

In some aspects of these embodiments, each R⁴ is heterocyclyl (e.g.,3-tetrahydrofuranyl) substituted with 0-3 occurrences of R⁵. In someaspects of these embodiments, each R⁴ is heterocyclyl (e.g.,3-tetrahydrofuranyl) substituted with 0 occurrences of R⁵.

In some aspects of these embodiments, each R⁴ is heterocyclylalkyl(e.g., 2-tetrahydrofuranylmethyl) substituted with 0-3 occurrences ofR⁵. In some aspects of these embodiments, each R⁴ is heterocyclylalkyl(e.g., 2-tetrahydrofuranylmethyl) substituted with 0 occurrences of R⁵.In some aspects of these embodiments, each R⁴ is heterocyclylalkylsubstituted with 0 occurrences of R⁵ and is represented by the followingstructure:

In some aspects of these embodiments, each R⁴ is cycloalkyl (e.g.,cyclopentyl) substituted with 0-3 occurrences of R⁵. In some aspects ofthese embodiments, each R⁴ is cycloalkyl (e.g., cyclopentyl) substitutedwith 0 occurrences of R⁵.

In some aspects of these embodiments, each R⁴ is cycloalkylalkyl (e.g.,cyclopropylmethyl) substituted with 0-3 occurrences of R⁵. In someaspects of these embodiments, each R⁴ is cycloalkylalkyl (e.g.,cyclopropylmethyl) substituted with 0 occurrences of R⁵.

In some aspects of these embodiments, each R⁴ is C₁₋₆ alkenyl (e.g.,ethenyl) substituted with 0-3 occurrences of R⁵. In some aspects ofthese embodiments, each R⁴ is C₁₋₆ alkenyl (e.g., ethenyl) substitutedwith one occurrence of R⁵. In some further aspect of these embodiments,each R⁵ is heteroaryl (e.g., 2-pyridinyl).

In some aspects of these embodiments, one R⁴ is C₁₋₆ alkyl (e.g.,methyl) substituted with 0 occurrences of R⁵ and the other R⁴ isheteroaralkyl (e.g., 3-indolylmethyl) substituted with one occurrence ofR⁵, wherein R⁵ is C₁₋₆ alkyl (e.g., methyl).

In some aspects of these embodiments, one R⁴ is C₁₋₆ alkyl (e.g.,methyl) substituted with 0 occurrences of R⁵ and the other R⁴ isheteroaralkyl (e.g., 3-indolylmethyl) substituted with one occurrence ofR⁵, wherein R⁵ is C₁₋₆ alkyl (e.g., methyl).

In some aspects of these embodiments, one R⁴ is C₁₋₆ alkyl (e.g.,methyl) and the other R⁴ is heteroaralkyl (e.g., 2-pyridinylmethyl),each of which is substituted with 0 occurrences of R⁵.

In some aspects of these embodiments, one R⁴ is heteroaralkyl (e.g.,2-pyridinylmethyl) substituted with 0 occurrences of R⁵ and the other R⁴is aralkyl (e.g., benzyl) substituted with one occurrence of R⁵, whereinR⁵ is C₁₋₆ alkoxy (e.g., methoxy).

In some aspects of these embodiments, one R⁴ is C₁₋₆ alkyl (e.g.,methyl) substituted with 0 occurrences of R⁵ and the other R⁴ is aralkyl(e.g., benzyl) substituted with one occurrence of R⁵, wherein R⁵ is C₁₋₆alkoxy (e.g., methoxy).

In some embodiments, R² is —NH₂ and R¹ is —N(R³)—C(O)—R⁴, wherein R³ ishydrogen and R⁴ is heteroaralkyl (e.g., 2-pyridinylmethyl) substitutedwith 0 occurrences of R⁵.

In some embodiments, each R⁶ is H.

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (II):

wherein R¹, R², R³, R⁴, R⁵, R⁶, o, p, m, n and X are as defined inFormula (I).

In some embodiments, a compound of Formula (I) or (II) is represented bya compound of Formula (IIa):

wherein R¹, R², R³, R⁴, R⁵, o, p and X are as defined in Formula (I).

In some embodiments, a compound of Formula (I), (II) or (IIa) isrepresented by a compound of Formula (III):

wherein R⁴, R⁵ and X are as defined in Formula (I).

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (IV):

wherein R¹, R², R³, R⁴ and R⁵ are as defined in Formula (I) and q is 0,1, 2, 3 or 4.

In some embodiments, a compound of Formula (I) or (IV) is represented bya compound of Formula (IVa):

wherein R⁴, R⁵ and q are as defined in Formula (IV).

In some embodiments, a compound of Formula (I) or (IV) is represented bya compound of Formula (IVb):

wherein R⁴, R⁵ and q are as defined in Formula (IV).

In some embodiments, a compound of Formula (I) or (IV) is represented bya compound of Formula (IVc):

wherein R⁴, R⁵ and q are as defined in Formula (IV).

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (V):

wherein R⁴ is C₁₋₆ alkyl, aralkyl or heteroaralkyl substituted with 0, 1or 2 occurrences of R⁵, wherein R⁵ is selected from methyl, methoxy,—NH₂, —N(CH₃)₂, —SO₂—CH₃, —NHC(O)CH₃, NO₂, CN, bromo or fluoro. In someembodiments of Formula (V), each R⁴ is the same. In some embodiments ofFormula (V), each R⁴ is different.

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (Va):

wherein R⁴ is C₁₋₆ alkyl, aralkyl or heteroaralkyl substituted with 0, 1or 2 occurrences of R⁵, wherein R⁵ is selected from methyl, methoxy,—NH₂, —N(CH₃)₂, —SO₂—CH₃, —NHC(O)CH₃, NO₂, CN, bromo or fluoro. In someembodiments of Formula (Va), each R⁴ is the same. In some embodiments ofFormula (Va), each R⁴ is different.

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (Vb):

wherein R⁴ is C₁₋₆ alkyl, aralkyl or heteroaralkyl substituted with 0, 1or 2 occurrences of R⁵, wherein R⁵ is selected from methyl, methoxy,—NH₂, —N(CH₃)₂, —SO₂—CH₃, —NHC(O)CH₃, NO₂, CN, bromo or fluoro. In someembodiments of Formula (Vb), each R⁴ is the same. In some embodiments ofFormula (Vb), each R⁴ is different.

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (VI):

wherein R⁴ is aralkyl substituted with 1 occurrence of R⁵, wherein R⁵ ismethoxy. In some aspects of these embodiments, each R⁴ is the same.

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (VIa):

wherein R⁴ is aralkyl substituted with 1 occurrence of R⁵, wherein R⁵ ismethoxy. In some aspects of these embodiments, each R⁴ is the same.

In some embodiments, a compound of Formula (I) is represented by acompound of Formula (VIb):

wherein R⁴ is aralkyl substituted with 1 occurrence of R⁵, wherein R⁵ ismethoxy. In some aspects of these embodiments, each R⁴ is the same.

In certain embodiments, exemplary compounds of Formula I include thecompounds described in Table 1 and in the Examples. A compound describedherein may be tested for its ability to inhibit glutaminase, e.g., by anassay as described in the Examples. For simplicity, the inhibitionactivity of these compounds is represented as an IC₅₀ tested in an assayof Example A or Example B in Table 1. Exemplary compounds are shown inTable 1 below. As shown, “A” refers to an inhibitor of glutaminase withan IC₅₀<100 nM. “B” refers to inhibitor of glutaminase with an IC₅₀between 100 nM and 500 nM. “C” refers to inhibitor of glutaminase withan IC₅₀ between 500 nM and 1000 nM. “D” refers to inhibitor ofglutaminase with an IC₅₀ between 1 μM and 2 μM. “E” refers to inhibitorof glutaminase with an IC₅₀ between 2 μM and 10 μM. “N/A” refers tocompounds wherein the IC₅₀ is unavailable.

TABLE 1 Cmpd # Structure IC₅₀ 1

B 2

C 3

C 4

E 5

B 6

B 7

E 8

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A

The compounds described herein can be made using a variety of synthetictechniques such as those described in the examples provided herein. Ascan be appreciated by the skilled artisan, methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds provided herein may contain one or more asymmetric centersand thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of these compounds are expressly included within the scope. Unlessotherwise indicated when a compound is named or depicted by a structurewithout specifying the stereochemistry and has one or more chiralcenters, it is understood to represent all possible stereoisomers of thecompound. The compounds provided herewith may also contain linkages(e.g., carbon-carbon bonds) or substituents that can restrict bondrotation, e.g. restriction resulting from the presence of a ring ordouble bond. Accordingly, all cis/trans and E/Z isomers are expresslyincluded.

The compounds provided herein (e.g. of Formula I) may also comprise oneor more isotopic substitutions. For example, H may be in any isotopicform, including ¹H, ²H (D or deuterium), and ³H (T or tritium); C may bein any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in anyisotopic form, including ¹⁶O and ¹⁸O; and the like. The compoundsprovided herein may also be represented in multiple tautomeric forms, insuch instances, expressly includes all tautomeric forms of the compoundsdescribed herein, even though only a single tautomeric form may berepresented (e.g., alkylation of a ring system may result in alkylationat multiple sites; all such reaction products are expressly included).All such isomeric forms of such compounds are expressly included. Allcrystal forms of the compounds described herein are expressly included.

The compounds provided herein include the compounds themselves, as wellas their salts and their prodrugs, if applicable. A salt, for example,can be formed between an anion and a positively charged substituent(e.g., amino) on a compound described herein. Suitable anions includechloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt canalso be formed between a cation and a negatively charged substituent(e.g., carboxylate) on a compound described herein. Suitable cationsinclude sodium ion, potassium ion, magnesium ion, calcium ion, and anammonium cation such as tetramethylammonium ion. Examples of prodrugsinclude esters and other pharmaceutically acceptable derivatives, which,upon administration to a subject, are capable of providing activecompounds.

The compounds provided herein may be modified by appending appropriatefunctionalities to enhance selected biological properties, e.g.,targeting to a particular tissue. Such modifications are known in theart and include those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

In an alternate embodiment, the compounds described herein may be usedas platforms or scaffolds that may be utilized in combinatorialchemistry techniques for preparation of derivatives and/or chemicallibraries of compounds. Such derivatives and libraries of compounds havebiological activity and are useful for identifying and designingcompounds possessing a particular activity. Combinatorial techniquessuitable for utilizing the compounds described herein are known in theart as exemplified by Obrecht, D. and Villalgrodo, J. M.,Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries, Pergamon-Elsevier ScienceLimited (1998), and include those such as the “split and pool” or“parallel” synthesis techniques, solid-phase and solution-phasetechniques, and encoding techniques (see, for example, Czarnik, A. W.,Curr. Opin. Chem. Bio., (1997) 1, 60. Thus, one embodiment relates to amethod of using the compounds described herein for generatingderivatives or chemical libraries comprising: 1) providing a bodycomprising a plurality of wells; 2) providing one or more compoundsidentified by methods described herein in each well; 3) providing anadditional one or more chemicals in each well; 4) isolating theresulting one or more products from each well. An alternate embodimentrelates to a method of using the compounds described herein forgenerating derivatives or chemical libraries comprising: 1) providingone or more compounds described herein attached to a solid support; 2)treating the one or more compounds identified by methods describedherein attached to a solid support with one or more additionalchemicals; 3) isolating the resulting one or more products from thesolid support. In the methods described above, “tags” or identifier orlabeling moieties may be attached to and/or detached from the compoundsdescribed herein or their derivatives, to facilitate tracking,identification or isolation of the desired products or theirintermediates. Such moieties are known in the art. The chemicals used inthe aforementioned methods may include, for example, solvents, reagents,catalysts, protecting group and deprotecting group reagents and thelike. Examples of such chemicals are those that appear in the varioussynthetic and protecting group chemistry texts and treatises referencedherein.

DEFINITIONS

The term “halo” or “halogen” refers to any radical of fluorine,chlorine, bromine or iodine.

The term “alkyl” refers to a saturated or unsaturated hydrocarbon chainthat may be a straight chain or branched chain, containing the indicatednumber of carbon atoms. For example, C₁-C₁₂ alkyl indicates that thegroup may have from 1 to 12 (inclusive) carbon atoms in it. The term“alkyl” includes alkenyl moieties. The term “haloalkyl” refers to analkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). The terms “arylalkyl” or “aralkyl” refer toan alkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “arylalkyl” or “aralkyl”include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl,and trityl groups.

The term “alkylene” or “cycloalkylene” refers to a divalent alkyl orcycloalkyl, e.g., —CH₂—, —CH₂CH₂—, and —CH₂CH₂CH₂—.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-12 carbon atoms and having one or more double bonds.Examples of alkenyl groups include, but are not limited to, allyl,propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the doublebond carbons may optionally be the point of attachment of the alkenylsubstituent.

The term “alkoxy” refers to an —O-alkyl radical. The term “haloalkoxy”refers to an alkoxy in which one or more hydrogen atoms are replaced byhalo, and includes alkoxy moieties in which all hydrogens have beenreplaced by halo (e.g., perfluoroalkoxy).

The term “aryl” refers to a monocyclic, bicyclic, or tricyclic aromatichydrocarbon ring system, wherein any ring atom capable of substitutioncan be substituted (e.g., by one or more substituents). Examples of arylmoieties include, but are not limited to, phenyl, naphthyl, andanthracenyl. Unless otherwise specified, any ring atom in an aryl can besubstituted by one or more substituents.

The term “cycloalkyl” as employed herein includes cyclic, bicyclic,tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12carbons. Any substitutable ring atom can be substituted (e.g., by one ormore substituents). The cycloalkyl groups can contain fused or spirorings. Fused rings are rings that share a common carbon atom. Examplesof cycloalkyl moieties include, but are not limited to, cyclopropyl,cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl.

The term “cycloalkylalkyl” as employed herein refers to an alkyl groupsubstituted with a cycloalkyl group.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to14-membered non-aromatic ring structures (e.g., 3- to 14-membered rings,more preferably 3- to 7-membered rings), whose ring structures includeone to four heteroatoms independently selected from O, N and S. Theheterocyclyl or heterocyclic groups can contain fused or spiro rings.Heterocycles can also be polycycles, with each group having, e.g., 5-7ring members. The term “heterocyclyl” or “heterocyclic group” includessaturated and partially saturated heterocyclyl structures. Theheteroatom may optionally be the point of attachment of the heterocyclylsubstituent.

The term “heteroaryl” refers to a 5-14 membered (i.e., a 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic)aromatic ring system having 1-3 ring heteroatoms if monocyclic, 1-6 ringheteroatoms if bicyclic, or 1-9 ring heteroatoms if tricyclic, said ringheteroatoms independently selected from O, N, and S (e.g., 1-3, 1-6, or1-9 ring heteroatoms of N, O, or S if monocyclic, bicyclic, ortricyclic, respectively). Any substitutable ring atom can be substituted(e.g., by one or more substituents).

Bicyclic and tricyclic ring systems containing one or more heteroatomsand both aromatic and non-aromatic rings wherein the point of attachmentfrom the ring system to the rest of the molecule is through anon-aromatic ring are considered to be heterocyclyl groups. Bicyclic ortricyclic ring systems where an aryl or a heteroaryl is fused to acycloalkyl or heterocyclyl and the point of attachment from the ringsystem to the rest of the molecule is through an aromatic ring areconsidered to be aryl or heteroaryl groups.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a heteroaryl group. The ring heteroatoms ofthe compounds provided herein include N—O, S(O), and S(O)₂.

Aryl, heteroaryl, cycloalkyl, and heterocyclyl groups, either alone or apart of a group (e.g., the aryl portion of an aralkyl group), areoptionally substituted at one or more substitutable atoms with, unlessspecified otherwise, substituents independently selected from: halo,—C≡N, C₁-C₄ alkyl, ═O, —OR^(b), —OR^(b′), —SR^(b), —SR^(b′), —(C₁-C₄alkyl)-N(R^(b))(R^(b)), —(C₁-C₄ alkyl)-N(R^(b))(R^(b′)),—N(R^(b))(R^(b)), —N(R^(b))(R^(b′)), —O—(C₁-C₄ alkyl)-N(R^(b))(R^(b)),—O—(C₁-C₄ alkyl)-N(R^(b))(R^(b′)), —(C₁-C₄ alkyl)-O—(C₁-C₄alkyl)-N(R^(b))(R^(b)), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R^(b))(R^(b′)),—C(O)—N(R^(b))(R^(b)), —(C₁-C₄ alkyl)-C(O)—N(R^(b))(R^(b)), —(C₁-C₄alkyl)-C(O)—N(R^(b))(R^(b′)), —OR^(b′), R^(b′), —C(O)(C₁-C₄ alkyl),—C(O)R^(b′), —C(O)N(R^(b′))(R^(b)), —N(R^(b))C(O)(R^(b)),—N(R^(b))C(O)(R^(b′)), —N(R^(b))SO₂(R^(b)), —SO₂N(R^(b))(R^(b)),—N(R^(b))SO₂(R^(b′)), and —SO₂N(R^(b))(R^(b′)), wherein any alkylsubstituent is optionally further substituted with one or more of —OH,—O—(C₁-C₄ alkyl), halo, —NH₂, —NH(C₁-C₄ alkyl), or —N(C₁-C₄ alkyl)₂;

-   -   each R^(b) is independently selected from hydrogen, and —C₁-C₄        alkyl; or    -   two R^(b)s are taken together with the nitrogen atom to which        they are bound to form a 4- to 8-membered heterocyclyl        optionally comprising one additional heteroatom selected from N,        S, and O; and    -   each R^(b′) is independently selected from C₃-C₇ carbocylyl,        phenyl, heteroaryl, and heterocyclyl, wherein one or more        substitutable positions on said phenyl, cycloalkyl, heteroaryl        or heterocycle substituent is optionally further substituted        with one or more of —(C₁-C₄ alkyl), —(C₁-C₄ fluoroalkyl), —OH,        —O—(C₁-C₄ alkyl), —O—(C₁-C₄ fluoroalkyl), halo, —NH₂, —NH(C₁-C₄        alkyl), or —N(C₁-C₄ alkyl)₂.

Heterocyclyl groups, either alone or as part of a group, are optionallysubstituted on one or more any substitutable nitrogen atom with oxo(═O), —C₁-C₄ alkyl, or fluoro-substituted C₁-C₄ alkyl.

The term “substituted” refers to the replacement of a hydrogen atom byanother group.

The term “selective” is meant at least 2-fold, 3-fold, 4-fold, 5-fold,6-fold, or 10-fold greater inhibition of glutaminase than other targets.

The term “inhibitor” as used herein means an agent that measurablyslows, stops, decreases or inactivates the enzymatic activity ofglutaminase to decrease to a level that is less than the glutaminasenormal levels of activity Inhibitors of glutaminase may be peptides ornucleic acids (e.g., glutamate). An agent can be evaluated to determineif it is an inhibitor by measuring either directly or indirectly theactivity of glutaminase when subjected to the agent. The activity of theagent can be measured, for example, against a control substance. In someinstances, the activity measured of the agent is for inhibition ofglutaminase.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, or a value, e.g., a numericalvalue, by “directly acquiring” or “indirectly acquiring” the physicalentity or value. “Directly acquiring” means performing a process (e.g.,performing a synthetic or analytical method) to obtain the physicalentity or value. “Indirectly acquiring” refers to receiving the physicalentity or value from another party or source (e.g., a third partylaboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Exemplary changes include making a physical entity from two ormore starting materials, shearing or fragmenting a substance, separatingor purifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

“Acquiring a sample” as the term is used herein, refers to obtainingpossession of a sample, e.g., a tissue sample or nucleic acid sample, by“directly acquiring” or “indirectly acquiring” the sample. “Directlyacquiring a sample” means performing a process (e.g., performing aphysical method such as a surgery or extraction) to obtain the sample.“Indirectly acquiring a sample” refers to receiving the sample fromanother party or source (e.g., a third party laboratory that directlyacquired the sample). Directly acquiring a sample includes performing aprocess that includes a physical change in a physical substance, e.g., astarting material, such as a tissue, e.g., a tissue in a human patientor a tissue that has was previously isolated from a patient. Exemplarychanges include making a physical entity from a starting material,dissecting or scraping a tissue; separating or purifying a substance(e.g., a sample tissue or a nucleic acid sample); combining two or moreseparate entities into a mixture; performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. Directlyacquiring a sample includes performing a process that includes aphysical change in a sample or another substance, e.g., as describedabove.

As used herein a “low” of E-cadherin expression compared to a referencestandard refers a low, decreased, or absent level of E-cadherinexpression compared to the level of E-cadherin expression in anepithelial cell as characterized by methods known in the art, e.g., onany one of the following references: (Yauch et al., (2005) Clin CancerRes 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thieryet al., (2002) Nature Reviews Cancer 2(6):442).

As used herein, a “high” level of vimentin compared to a referencestandard refers to a high or increased level of vimentin expressioncompared to the level of expression of vimentin in an epithelial cell ascharacterized by methods known in the art, e.g., on any one of thefollowing references: (Yauch et al., (2005) Clin Cancer Res 11:24;Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al.,(2002) Nature Reviews Cancer 2(6):442).

As used herein, a “low” or “decreased”, level of pyruvate carboxylaseexpression compared to a reference standard refers to a low, decreased,or absent level of E-cadherin expression compared to the level ofE-cadherin expression in an epithelial cell as characterized by methodsknown in the art, e.g., on any one of the following references: (Yauchet al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol.21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer2(6):442).

As used herein, “cancer” and “tumor” are synonymous terms. The term“cancer” or “tumor” refer to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Cancer cells are often in the form of a tumor, but such cells may existalone within an animal, or may be a non-tumorigenic cancer cell, such asa leukemia cell. The cells can possess characteristics typical of amesenchymal cell, such as characterized on any one of the followingreferences: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner etal., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) NatureReviews Cancer 2(6):442).

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Methods of Evaluating Compounds

Glutaminase activity can be monitored by detecting production of eitherof the products of the reaction, glutamate or ammonia. In someembodiments, glutamate production is measured because ammonia is aproduct of any of a number of biological reactions.

Glutamate production can be measured by any of a number of standardmethods known in the art, e.g., chemical and chromatographic detectionmethods and coupled enzyme assays that utilize NADH and glutamatedehydrogenase. Extracellular glutamate concentrations can also bemeasured in vivo, using microdialysis methods known in the art. Onesuitable method for measuring glutamate is a microtiter-based two-stepassay in which glutamate formed in the initial step is quantitativelydeaminated by glutamate dehydrogenase to yield an equivalent amount ofNADH (Godfrey et al., 1977; Kvamme et al., 1985), which can then bedetected spectrophotometrically.

Methods of Treatment

In one embodiment, provided is a method for treating or preventing adisease, condition or disorder as described herein (e.g., treating)comprising administering a compound, a pharmaceutically acceptable saltof a compound or pharmaceutical composition comprising a compounddescribed herein (e.g., a compound of formula (I) or in Table 1).

The compounds and compositions described herein can be administered tocells in culture, e.g. in vitro or ex vivo, or to a subject, e.g., invivo, to treat, prevent, and/or diagnose a variety of disorders,including those described herein below.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of a compound, alone or in combinationwith, a second compound to a subject, e.g., a patient, or application oradministration of the compound to an isolated tissue or cell, e.g., cellline, from a subject, e.g., a patient, who has a disorder (e.g., adisorder as described herein), a symptom of a disorder, or apredisposition toward a disorder, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisorder, one or more symptoms of the disorder or the predispositiontoward the disorder (e.g., to prevent at least one symptom of thedisorder or to delay onset of at least one symptom of the disorder).

As used herein, an amount of a compound effective to treat a disorder,or a “therapeutically effective amount” refers to an amount of thecompound which is effective, upon single or multiple dose administrationto a subject, in treating a cell, or in curing, alleviating, relievingor improving a subject with a disorder beyond that expected in theabsence of such treatment.

As used herein, an amount of a compound effective to prevent a disorder,or a “a prophylactically effective amount” of the compound refers to anamount effective, upon single- or multiple-dose administration to thesubject, in preventing or delaying the occurrence of the onset orrecurrence of a disorder or a symptom of the disorder.

The term “patient” and “subject” are synonymous, and as used herein,refer to an animal, typically a human (i.e., a male or female of any agegroup, e.g., a pediatric patient or adult patient or other mammal, suchas primates (e.g., cynomolgus monkeys, rhesus monkeys); commerciallyrelevant mammals such as cattle, pigs, horses, sheep, goats, cats,and/or dogs; and/or birds, including commercially relevant birds such aschickens, ducks, geese, and/or turkeys, that will be or has been theobject of treatment, observation, and/or experiment. When the term isused in conjunction with administration of a compound or drug, then thepatient has been the object of treatment, observation, and/oradministration of the compound or drug.

Cancers

The methods described herein can be used with any cancer, for examplethose described by the National Cancer Institute. A cancer can beevaluated to determine whether it is using a method described herein.Exemplary cancers can include but are not limited to, lung cancer, e.g.,non-small cell lung cancer; breast cancer; or hepatocellular carcinoma,osteosarcoma, lipomas, chondrosarcoma, or mesothelioma.

The cancer can be a primary tumor, i.e., located at the anatomical siteof tumor growth initiation. The cancer can also be metastatic, i.e.,appearing at least a second anatomical site other than the anatomicalsite of tumor growth initiation. The cancer can be a recurrent cancer,i.e., cancer that returns following treatment, and after a period oftime in which the cancer was undetectable. The recurrent cancer can beanatomically located locally to the original tumor, e.g., anatomicallynear the original tumor; regionally to the original tumor, e.g., in alymph node located near the original tumor; or distantly to the originaltumor, e.g., anatomically in a region remote from the original tumor.

Cancer Combination therapies

In some embodiments, a compound described herein is administeredtogether with one or more additional cancer treatments. Exemplary cancertreatments include, for example: chemotherapy, targeted therapies suchas antibody therapies, immunotherapy, and hormonal therapy. Examples ofeach of these treatments are provided below.

Chemotherapy

In some embodiments, a compound described herein is administered withone or more chemotherapies. Chemotherapy is the treatment of cancer withdrugs that can destroy cancer cells. “Chemotherapy” usually refers tocytotoxic drugs which affect rapidly dividing cells in general, incontrast with targeted therapy. Chemotherapy drugs interfere with celldivision in various possible ways, e.g., with the duplication of DNA orthe separation of newly formed chromosomes. Most forms of chemotherapytarget all rapidly dividing cells and are not specific for cancer cells,although some degree of specificity may come from the inability of manycancer cells to repair DNA damage, while normal cells generally can.

Examples of chemotherapeutic agents used in cancer therapy include, forexample, antimetabolites (e.g., folic acid, purine, and pyrimidinederivatives) and alkylating agents (e.g., nitrogen mustards,nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes,aziridines, spindle poison, cytotoxic agents, toposimerase inhibitorsand others). Exemplary agents include Aclarubicin, Actinomycin,Alitretinon, Altretamine, Aminopterin, Aminolevulinic acid, Amrubicin,Amsacrine, Anagrelide, Arsenic trioxide, Asparaginase, Atrasentan,Belotecan, Bexarotene, endamustine, Bleomycin, Bortezomib, Busulfan,Camptothecin, Capecitabine, Carboplatin, Carboquone, Carmofur,Carmustine, Celecoxib, Chlorambucil, Chlormethine, Cisplatin,Cladribine, Clofarabine, Crisantaspase, Cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Daunorubicin, Decitabine, Demecolcine,Docetaxel, Doxorubicin, Efaproxiral, Elesclomol, Elsamitrucin,Enocitabine, Epirubicin, Estramustine, Etoglucid, Etoposide,Floxuridine, Fludarabine, Fluorouracil (5FU), Fotemustine, Gemcitabine,Gliadel implants, Hydroxycarbamide, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Irofulven, Ixabepilone, Larotaxel, Leucovorin, Liposomaldoxorubicin, Liposomal daunorubicin, Lonidamine, Lomustine, Lucanthone,Mannosulfan, Masoprocol, Melphalan, Mercaptopurine, Mesna, Methotrexate,Methyl aminolevulinate, Mitobronitol, Mitoguazone, Mitotane, Mitomycin,Mitoxantrone, Nedaplatin, Nimustine, Oblimersen, Omacetaxine, Ortataxel,Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Pentostatin,Pirarubicin, Pixantrone, Plicamycin, Porfimer sodium, Prednimustine,Procarbazine, Raltitrexed, Ranimustine, Rubitecan, Sapacitabine,Semustine, Sitimagene ceradenovec, Satraplatin, Streptozocin,Talaporfin, Tegafur-uracil, Temoporfin, Temozolomide, Teniposide,Tesetaxel, Testolactone, Tetranitrate, Thiotepa, Tiazofurin, Tioguanine,Tipifarnib, Topotecan, Trabectedin, Triaziquone, Triethylenemelamine,Triplatin, Tretinoin, Treosulfan, Trofosfamide, Uramustine, Valrubicin,Verteporfin, Vinblastine, Vincristine, Vindesine, Vinflunine,Vinorelbine, Vorinostat, Zorubicin, and other cytostatic or cytotoxicagents described herein.

Because some drugs work better together than alone, two or more drugsare often given at the same time. Often, two or more chemotherapy agentsare used as combination chemotherapy. In some embodiments, thechemotherapy agents (including combination chemotherapy) can be used incombination with a compound described herein.

Targeted Therapy

In some embodiments, a compound described herein is administered withone or more targeted therapies. Targeted therapy constitutes the use ofagents specific for the deregulated proteins of cancer cells. Smallmolecule targeted therapy drugs are generally inhibitors of enzymaticdomains on mutated, overexpressed, or otherwise critical proteins withinthe cancer cell. Prominent examples are the tyrosine kinase inhibitorssuch as Axitinib, Bosutinib, Cediranib, dasatinib, erlotinib, imatinib,gefitinib, lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib,Sunitinib, and Vandetanib, and also cyclin-dependent kinase inhibitorssuch as Alvocidib and Seliciclib. Monoclonal antibody therapy is anotherstrategy in which the therapeutic agent is an antibody whichspecifically binds to a protein on the surface of the cancer cells.Examples include the anti-HER2/neu antibody trastuzumab (HERCEPTIN®)typically used in breast cancer, and the anti-CD20 antibody rituximaband Tositumomab typically used in a variety of B-cell malignancies.Other exemplary antibodies include Cetuximab, Panitumumab, Trastuzumab,Alemtuzumab, Bevacizumab, Edrecolomab, and Gemtuzumab. Exemplary fusionproteins include Aflibercept and Denileukin diftitox. In someembodiments, the targeted therapy can be used in combination with acompound described herein.

Exemplary additional therapeutic agents can also include epidermalgrowth factor receptor (EGFR) inhibitors, e.g., cetuximab, panitumumab,gefitinib, erlotinib, nimotuzamab, matuzamab, zalutumumab, or lapatinib.Resistance to EGFR inhibitors can occur as a result of the transition ofa cell to a mesenchymal phenotype or a mesenchymal phenotype, and tumorswith EGFR mutations and mesenchymal phenotype can be less sensitive toEGFR inhibitors (see for example, Sequist et al., (2011) Sci Transl Med.3:75. Buck et al., (2007) Mol Cancer Ther. 6: 532; Thomson et al.,(2008) Clin Exp Metastasis 25: 843).

Targeted therapy can also involve small peptides as “homing devices”which can bind to cell surface receptors or affected extracellularmatrix surrounding the tumor. Radionuclides which are attached to thesepeptides (e.g., RGDs) eventually kill the cancer cell if the nuclidedecays in the vicinity of the cell. An example of such therapy includesBEXXAR®.

Immunotherapy

In some embodiments, a compound described herein is administered withone or more immunotherapies. Cancer immunotherapy refers to a diverseset of therapeutic strategies designed to induce the patient's ownimmune system to fight the tumor. Contemporary methods for generating animmune response against tumors include intravesicular BCG immunotherapyfor superficial bladder cancer, and use of interferons and othercytokines to induce an immune response in renal cell carcinoma andmelanoma patients.

Allogeneic hematopoietic stem cell transplantation can be considered aform of immunotherapy, since the donor's immune cells will often attackthe tumor in a graft-versus-tumor effect. In some embodiments, theimmunotherapy agents can be used in combination with a compounddescribed herein.

Hormonal Therapy

In some embodiments, a compound described herein is administered withone or more hormonal therapies. The growth of some cancers can beinhibited by providing or blocking certain hormones. Common examples ofhormone-sensitive tumors include certain types of breast and prostatecancers. Removing or blocking estrogen or testosterone is often animportant additional treatment. In certain cancers, administration ofhormone agonists, such as progestogens may be therapeuticallybeneficial. In some embodiments, the hormonal therapy agents can be usedin combination with a compound described herein.

Neuronal Disorders

A compound or composition described herein can be used to treat orprevent neuronal cell death as a result of an injury to neuronal tissue,e.g., nervous tissue exposed to an ischemic or hypoxic event, to traumaor to a chronic neurodegenerative disorder. A “neuronal disorder” is aneurological disease or disorder that is associated with glutamateexcitotoxicity, e.g., cerebral ischemia or hypoxia resulting from anneurological event such as a stroke or ischemic event. Treatment withthe compound may be in an amount effective to provide a neuroprotectiveeffect, e.g., to prevent neuronal cell death.

Compositions and Routes of Administration

The compositions delineated herein include the compounds delineatedherein (e.g., a compound described herein), as well as additionaltherapeutic agents if present, in amounts effective for achieving amodulation of disease or disease symptoms, including those describedherein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a subject, together witha compound provided herewith, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions provided herewith include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutical compositions provided herewith may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions provided herewith may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions provided herewith may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions provided herewith may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound providedherewith with a suitable non-irritating excipient which is solid at roomtemperature but liquid at the rectal temperature and therefore will meltin the rectum to release the active components. Such materials include,but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions providedherewith is useful when the desired treatment involves areas or organsreadily accessible by topical application. For application topically tothe skin, the pharmaceutical composition should be formulated with asuitable ointment containing the active components suspended ordissolved in a carrier. Carriers for topical administration of thecompounds provided herewith include, but are not limited to, mineraloil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions provided herewith may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded.

The pharmaceutical compositions provided herewith may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions provided herewith comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds provided herewith. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds providedherewith in a single composition.

The compounds described herein can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.5 toabout 100 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositionsprovided herewith will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination provided herewith may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

Patient Selection and Monitoring

The compounds described herein can inhibit glutaminase. Accordingly, apatient and/or subject can be selected for treatment using a compounddescribed herein by first evaluating the patient and/or subject todetermine whether the subject is in need of inhibition of glutaminase,and if the subject is determined to be in need of glutaminaseinhibition, then administering to the subject a compound describedherein.

A subject can be evaluated as being in need of glutaminase inhibitionusing methods known in the art, e.g., by measuring the presence and/oractivity of glutaminase in the patient. In some embodiments, theactivity and/or level of glutaminase is evaluated in the cancer.

A patient receiving a compound described herein can be monitored, forexample, for improvement in the condition and/or adverse effects.Improvement of a patient's condition can be evaluated, for example, bymonitoring the growth, absence of growth, or regression of the cancer(e.g., a tumor). In some embodiments, the patient is evaluated using aradiological assay or evaluation of hemolytic parameters.

A patient and/or subject can be selected for treatment using a compounddescribed hereby by optionally, acquiring a patient sample; evaluatingthe sample to determine whether the sample is characterized by i) a lowlevel of E-cadherin expression compared to a reference standard, ii) ahigh level of vimentin expression compared to a reference standard,and/or iii) a low or decreased level of pyruvate carboxylase expressioncompared to a reference standard; and if the patient is determined tohave a low level of E-cadherin expression compared to a referencestandard, or a high level of vimentin expression compared to a referencestandard, then the patient is administered a compound described herein.

In some embodiments, the level of E-cadherin expression is compared to areference standard, wherein the reference standard is the level ofE-cadherin expression in an epithelial cell as characterized on any oneof the following references: (Yauch et al., (2005) Clin Cancer Res11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery etal., (2002) Nature Reviews Cancer 2(6):442). In some embodiments, thelevel of E-cadherin expression is low, decreased, or absent compared tothe reference standard. In some embodiments, the level of E-cadherinexpression is measured by the evaluation of the level of RNA thatencodes E-cadherin. In some embodiments, the level of E-cadherinexpression is evaluated by the level of E-cadherin protein expression.In some embodiments the level of E-cadherin expression is at least 5,10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90% less than the referencestandard. In some embodiments the level of E-cadherin expression is atleast a 1.5, 2, 5, 10, 15, 20, 25, 50, 75, 100 fold decrease inexpression compared to the reference standard.

In some embodiments, the level of vimentin expression is compared to areference standard, wherein the reference standard is the level ofvimentin expression in an epithelial cell as characterized on any one ofthe following references: (Yauch et al., (2005) Clin Cancer Res 11:24;Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al.,(2002) Nature Reviews Cancer 2(6):442). In some embodiments, the levelof vimentin expression is measured by the evaluation of the level of RNAthat encodes vimentin. In some embodiments, the level of vimentinexpression is evaluated by the level of vimentin protein expression. Insome embodiments the level of vimentin expression is at least 5, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, or 90% greater than the referencestandard. In some embodiments the level of vimentin expression is atleast a 1.5, 2, 5, 10, 15, 20, 25, 50, 75, 100 fold increase inexpression compared to the reference standard.

In some embodiments, the level of pyruvate carboxylase expression is lowor decreased compared to a reference standard, wherein the referencestandard is the level of pyruvate carboxylase expression in anepithelial cell as characterized on any one of the following references:(Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) AnnOncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer2(6):442). In some embodiments, the level of pyruvate carboxylaseexpression is high or increased compared to the reference standard. Insome embodiments, the level of pyruvate carboxylase expression ismeasured by the evaluation of the level of RNA that encodes pyruvatecarboxylase. In some embodiments, the level of vimentin expression isevaluated by the level of pyruvate carboxylase protein expression. Insome embodiments the level of pyruvate carboxylase expression is atleast 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90% greater than thereference standard. In some embodiments the level of pyruvatecarboxylase expression is at least a 1.5, 2, 5, 10, 15, 20, 25, 50, 75,100 fold increase in expression compared to the reference standard.

Patient Sample

The terms “patient sample”, “subject sample”, and “sample” are usedinterchangeably herein. The patient sample can be a tissue, or bodilyfluid, or bodily product. Tissue samples can include fixed, paraffinembedded, fresh, or frozen samples. For example, the tissue sample caninclude a biopsy, cheek swab. Exemplary tissues include lung, breast,brain, nervous tissue, kidney, ovary, thyroid, pancreas, colon,prostate, lymph node, skin, hair follicles and nails. Exemplary samplesinclude samples derived from solid tumors. Exemplary bodily fluidsinclude blood, plasma, urine, lymph, tears, sweat, saliva, semen, andcerebrospinal fluid. Exemplary bodily products include exhaled breath.

The tissue, fluid or product can be removed from the patient andanalyzed. The evaluation can include one or more of: performing theanalysis of the tissue, fluid or product; requesting analysis of thetissue fluid or product; requesting results from analysis of the tissue,fluid or product; or receiving the results from analysis of the tissue,fluid or product.

The sample tissue, fluid, or product can be analyzed for the expressionlevel of a gene described herein, e.g., E-cadherin, vimentin, pyruvatecarboxylase. The sample tissue, fluid, or product can be analyzed forthe expression level of a protein described herein, e.g., E-cadherin,vimentin, pyruvate carboxylase. The sample tissue, fluid or product canfurther be analyzed for the level of gene expression of a gene orplurality of genes of a preselected signaling pathway or phenotypicpathway, e.g., the epithelial to mesenchymal transition pathway,E-cadherin pathway, vimentin pathway, or the pyruvate carboxylasepathway. The sample tissue, fluid or product can further be analyzed forthe level of protein expression of a protein or plurality of proteins ofa preselected signaling pathway or phenotypic pathway, e.g., theepithelial to mesenchymal transition pathway, E-cadherin pathway,vimentin pathway, or the pyruvate carboxylase pathway.

Methods of Evaluating Samples

The expression level of a gene described herein, e.g., E-cadherin,vimentin, and pyruvate carboxylase, can be assessed using any of a widevariety of well known methods for detecting expression of a transcribedmolecule, gene, protein, mRNA, genomic DNA, or cDNA. Gene expression canbe measured or monitored by measure of a gene transcript, e.g., mRNA, bya measure of the quantity of a translated protein, or by a measure ofgene product activity; any of which can be measured using standardtechniques known to one of skill in the art. Non-limiting examples ofsuch methods include nucleic acid hybridization methods, nucleic acidreverse transcription methods, nucleic acid amplification methods,immunological methods for detection of proteins, protein purificationmethods, protein function or activity assays.

E-Cadherin

The E-cadherin gene is located on human chromosome 16. E-cadherin is aclassical cadherin of the cadherin superfamily. The encoded E-cadherinprotein is a calcium dependent cell-cell adhesion glycoprotein comprisedof five extracellular cadherin repeats, a transmembrane region and ahighly conserved cytoplasmic tail. Mutations in this gene have beencorrelated with cancer, including gastric, breast, colorectal, thyroidand ovarian cancers. Loss of function of E-cadherin is contemplated tocontribute to cancer progression by increasing proliferation, invasion,and/or metastasis. The ectodomain of this protein mediates bacterialadhesion to mammalian cells and the cytoplasmic domain is required forinternalization. Identified E-cadherin transcript variants arise frommutation at consensus splice sites.

Vimentin

The vimentin gene is located on human chromosome 10 and encodes a memberof the intermediate filament family of proteins. Intermediate filaments,along with microtubules and actin microfilaments, make up the cellularcytoskeleton, which helps maintain cell shape and integrity of thecytoplasm, as well as stabilizing cytoskeletal interactions. Vimentinalso functions in mediating immune responses, control of the transportof low-density lipoprotein derived cholesterol from lysosomes to thesites of esterification, and as an organizer of a number of criticalproteins involved in attachment, migration, and cell signaling.

Pyruvate Carboxylase (PC)

The PC gene is located on human chromosomes 11 and encodes the proteinpyruvate carboxylase, which catalyzes the carboxylation of pyruvate tooxaloacetate. The active enzyme is a homotetramer arranged in atetrahedron which is located exclusively in the mitochondrial matrix.Pyruvate carboxylase is involved in multiple cellular processesincluding gluconeogenesis, lipogenesis, insulin secretion and synthesisof the neurotransmitter glutamate. Mutations in this gene have beenassociated with pyruvate carboxylase deficiency. Alternatively splicedtranscript variants with different 5′ UTRs, but encoding the sameprotein, have been identified.

Nucleic Acid Molecules

The methods described herein can pertain to the evaluation of a samplefor the expression of a gene described herein, e.g., E-cadherin,vimentin, pyruvate carboxylase; based on isolated nucleic acids whichcorrespond to the gene described herein, e.g., the mRNA level ofE-cadherin; the mRNA level of vimentin; the mRNA level of pyruvatecarboxylase. As used herein, the term “nucleic acid” or “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. An “isolated” nucleic acid molecule can be freeof sequences (such as protein-encoding sequences) which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. An “isolated” nucleic acid molecule, such mRNA, can besubstantially free of other cellular material cellular material or othercontaminating proteins from the cell or tissue source from which thenucleic acid is derived.

A nucleic acid molecule described herein can be isolated using standardmolecular biology techniques and the sequence information available indatabase records known to those of skill in the art. Using all or aportion of such nucleic acid sequences, nucleic acid molecules describedherein can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., ed., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule described herein can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid molecules so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule can beprepared by standard synthetic techniques, e.g., using an automated DNAsynthesizer.

An isolated nucleic acid molecule can comprise a nucleic acid moleculewhich has a nucleotide sequence complementary to the nucleotide sequenceof a nucleic acid corresponding to gene described herein, or to thenucleotide sequence of a nucleic acid encoding a protein whichcorresponds to the gene described herein. A nucleic acid molecule whichis complementary to a given nucleotide sequence is one which issufficiently complementary to the given nucleotide sequence that it canhybridize to the given nucleotide sequence thereby forming a stableduplex.

A nucleic acid molecule described herein can comprise only a portion ofa nucleic acid sequence. Such nucleic acid molecules can be used, forexample, as a probe or primer. The probe/primer can be one or moresubstantially purified oligonucleotides. Probes based on the sequence ofa nucleic acid molecules described herein can be used to detecttranscripts or genomic sequences corresponding to the genes describedherein. The probe can contain comprise a label group, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as part of a diagnostic test kit for identifyingcells or tissues which express the protein, such as by measuring levelsof a nucleic acid molecule encoding the protein in a sample of cellsfrom a patient, e.g., detecting mRNA levels.

Methods for Detection of Gene Expression

Methods of detecting and/or quantifying a gene transcript, e.g., mRNA orcDNA made therefrom, can include but are not limited to Southern Blotanalysis, Northern Blot analysis, polymerase chain reaction (PCR)analyses and probe arrays. Methods of detecting and/or quantifying agene transcript, e.g., mRNA or cDNA made therefrom, can include but arenot limited to hybridization based methods, e.g., hybridization with aprobe that is specific for the gene transcript, e.g., mRNA or cDNA madetherefrom. The level of a gene transcript, e.g., mRNA or cDNA madetherefrom, can be assayed by applying the sample, or the mRNA or cDNAmade therefrom, or amplified from; to a nucleic acid microarray, or chiparray.

The level of a gene transcript, e.g., mRNA or cDNA made therefrom, canbe assayed by a polymerase chain reaction (PCR) based method, e.g.,quantitative PCR, quantitative real time PCR, real time PCR, reversetranscription PCR, real time reverse transcription PCR. The level of agene transcript, e.g., mRNA or cDNA made therefrom, can be assayed by asequencing based method, e.g., quantitative RNA sequencing.

The level of a gene transcript, e.g., mRNA, can be determined by in situor by in vitro methods known in the art. For in vitro methods, any RNAisolation technique that does not select against the isolation of mRNAcan be utilized for the purification of RNA from a sample, e.g., fromcells of a sample (see, e.g., Ausubel et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, New York 1987-1999). Additionally,large numbers of tissue samples can readily be processed usingtechniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155). For in situ methods, mRNA does not need to beisolated from the cells prior to detection. In such methods, a cell ortissue sample can be prepared/processed using known histologicalmethods. The sample can then be immobilized on a support, and thencontacted with a probe that can hybridize to mRNA that encodes the genetranscript of interest.

Determinations can be based on absolute expression level; normalizedexpression level, or relative expression level; of a gene transcript,e.g., mRNA. Expression levels can be normalized by correcting theabsolute expression level of a gene transcript by comparing itsexpression level to the expression level of another gene which is stablyexpressed, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such ashistone H3 gene or the actin gene. This normalization allows thecomparison of the expression level in one sample to another sample,e.g., a first sample taken from a patient to a second sample taken fromthe same patient, e.g., from another tissue or at a different timepoint; or between samples from different sources, e.g., a patient samplefrom one patient to a patient sample from another patient.

The expression level can be provided as a relative expression level. Therelative expression level can be determined by comparing the absolutelevel of expression of the gene transcript, e.g., mRNA, to a referencestandard. The reference standard can include the level of expression ofthe gene transcript of interest in a genotypically or phenotypicallydefined sample. The reference standard can be the level of expression ofthe gene transcript of interest, e.g., E-cadherin, vimentin, pyruvatecarboxylase, in a cell genotypically or phenotypically characterized asan epithelial cell. An epithelial cell can be characterized as in anyone of the following references: (Yauch et al., (2005) Clin Cancer Res11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery etal., (2002) Nature Reviews Cancer 2(6):442).

The expression level of a gene transcript described herein, e.g.,E-cadherin, vimentin, pyruvate carboxylase, can be measured at least attwo time-points to determine if a change in the level of expression hasoccurred. For example, the level of expression can be measured pre- andpost-treatment with a compound described herein, or at one or moretime-points while treatment with a compound described herein is ongoing.If the expression level is found to be decreased, e.g., decreasedexpression of E-cadherin compared to a reference standard and/orincreased expression of vimentin compared to a reference standard; thesubject may be administered treatment with a compound described herein.The reference standard can be the level of expression of the genetranscript of interest in an epithelial cell characterized. Anepithelial cell can be characterized by methods known in the art, e.g.,as in any one of the following references: (Yauch et al., (2005) ClinCancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89;Thiery et al., (2002) Nature Reviews Cancer 2(6):442).

Proteins

The methods described herein can pertain to the evaluation of a samplefor the expression of a gene described herein, e.g., E-cadherin,vimentin, pyruvate carboxylase; based on isolated proteins whichcorrespond to the gene described herein, e.g., the protein level ofE-cadherin; the protein level of vimentin; the protein level of pyruvatecarboxylase. This can also include the evaluation of biologically activeportions, variants, isoforms, or splice variants thereof. The nativepolypeptide corresponding to the protein of interest can be isolatedfrom the sample by an appropriate purification scheme using standardprotein purification techniques known to those of skill in the art.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived. The language “substantially free of cellularmaterial” includes preparations of protein in which the protein isseparated from cellular components of the cells from which it isisolated. Biologically active portions of a polypeptide can includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the protein, which includefewer amino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding protein.

Methods for Detection of Protein Expression

The level of expression of a protein or polypeptide can be detected andquantified by any of a number of means well known to those of skill inthe art. Methods of detecting and/or quantifying a protein orpolypeptide described herein, e.g., E-cadherin, vimentin, pyruvatecarboxylase; can include but are not limited to biochemical methods suchas electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, or various immunoassays such as fluid orgel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, Westernblotting, immunohistochemistry, in situ hybridization,fluorescence-activated cell sorting (FACS) and the like. A skilledartisan can readily adapt known protein/antibody detection methods foruse in determining whether cells express the protein or polypeptidedescribed herein.

A protein or polypeptide can be detected using an immunoassay. As usedherein, immunoassays include assays that utilize an antibody tospecifically bind to a protein or polypeptide. An immunoassay can becharacterized by the detection of specific binding of a protein orpolypeptide to an antibody as opposed to the use of other physical orchemical properties to isolate, target, and quantify the polypeptide.The polypeptide can be detected and/or quantified using any of a numberof well recognized immunological binding assays (see, e.g., U.S. Pat.Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review ofthe general immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th EditionImmunoassays for the detection and/or quantification of a protein orpolypeptide can take a wide variety of formats well known to those ofskill in the art.

An antibody capable of binding to a protein or polypeptide, e.g., anantibody with a detectable label (either directly or indirectlylabeled), corresponding to a protein or polypeptide described herein,e.g., E-cadherin, vimentin, pyruvate carboxylase, can be used to detectthe protein or polypeptide. Antibodies can be polyclonal or monoclonal.An intact antibody, or a fragment thereof, e.g., Fab or F(ab′)₂ can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling, i.e., physically linking a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The antibody can also be labeled, e.g., a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. Anantibody derivative, e.g., an antibody conjugated with a substrate orwith the protein or ligand of a protein-ligand pair, e.g.,biotin-streptavidin, or an antibody fragment, e.g., a single-chainantibody, an isolated antibody hypervariable domain, etc, which bindsspecifically with a protein described herein, e.g., E-cadherin,vimentin, pyruvate carboxylase, such as the protein encoded by the openreading frame corresponding to the gene transcript of a protein orpolypeptide described herein, e.g., E-cadherin, vimentin, pyruvatecarboxylase, or such a protein or polypeptide which has undergone all ora portion of its normal post-translational modification, is used.

Proteins from cells can be isolated using techniques that are well knownto those of skill in the art. The protein isolation methods employedcan, for example, be such as those described in Harlow and Lane (Harlowand Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

The expression level can be provided as a relative expression level. Therelative expression level can be determined by comparing the absolutelevel of expression of the protein, to a reference standard. Thereference standard can include the level of expression of the protein ofinterest in a genotypically or phenotypically defined sample. Thereference standard can be the level of expression of the protein ofinterest, e.g., E-cadherin, vimentin, pyruvate carboxylase, in a cellgenotypically or phenotypically characterized as an epithelial cell. Anepithelial cell can be characterized by methods known in the art, e.g.,as described in on any one of the following references: (Yauch et al.,(2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol.21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer2(6):442).

The expression level of a protein or polypeptide described herein, e.g.,E-cadherin, vimentin, pyruvate carboxylase, can be measured at least attwo time-points to determine if a change in the level of expression hasoccurred. For example, the level of expression can be measured pre- andpost-treatment with a compound described herein, or at one or moretime-points while treatment with a compound described herein is ongoing.If the expression level is found to be decreased, e.g., decreasedexpression of E-cadherin compared to a reference standard and/orincreased expression of vimentin compared to a reference standard; thesubject may be administered treatment with a compound described herein.

Kits

Also described herein are kits comprising a means to assay the level ofgene expression of a gene described herein, e.g., E-cadherin, vimentin,pyruvate carboxylase. For example, the kit can include an agent capableof interacting with a gene expression product of a gene describedherein, e.g., E-cadherin, vimentin, pyruvate carboxylase. The kit caninclude a plurality of agents capable of interacting with geneexpression products of a plurality of genes described herein, e.g.,E-cadherin, vimentin, pyruvate carboxylase. The agent can include, butis not limited to, an antibody, a plurality of antibodies, anoligonucleotide, or a plurality of oligonucleotides. The gene expressionproduct can include, but is not limited to, a transcribed molecule, aRNA molecule, a polypeptide, a protein, genomic DNA, or cDNA.

The kit can further optionally include reagents for performing theassays described herein. For example, the kit can include buffers,solvents, stabilizers, preservatives, purification columns, detectionreagents, and enzymes, which may be necessary for isolating nucleicacids from a patient sample, amplifying the samples, e.g., by qRT-PCR,and applying the samples to the agent described above; or for isolatingproteins from a subject sample, and applying the samples to the agentdescribed above; or reagents for directly applying the subject sample tothe agent described above. A kit can also include positive and negativecontrol samples, e.g., control nucleic acid samples (e.g., nucleic acidsample from a non-cancer subject, or a non-tumor tissue sample, or asubject who has not received treatment for cancer, or other test samplesfor testing at the same time as subject samples. A kit can also includeinstructional material, which may provide guidance for collecting andprocessing patient samples, applying the samples to the level of geneexpression assay, and for interpreting assay results.

The components of the kit can be provided in any form, e.g., liquid,dried, semi-dried, or in lyophilized form, or in a form for storage in afrozen condition. Typically, the components of the kit are provided in aform that is sterile. When reagents are provided in a liquid solution,the liquid solution generally is an aqueous solution, e.g., a sterileaqueous solution. When reagents are provided in a dried form,reconstitution generally is accomplished by the addition of a suitablesolvent. The solvent, e.g., sterile buffer, can optionally be providedin the kit.

The kit can include one or more containers for the kit components in aconcentration suitable for use in the level of gene expression assays orwith instructions for dilution for use in the assay. The kit can containseparate containers, dividers or compartments for the assay components,and the informational material. For example, the positive and negativecontrol samples can be contained in a bottle or vial, the clinicallycompatible classifier can be sealed in a sterile plastic wrapping, andthe informational material can be contained in a plastic sleeve orpacket. The kit can include a plurality (e.g., a pack) of individualcontainers, each containing one or more unit forms (e.g., for use withone assay) of an agent. The containers of the kits can be air tightand/or waterproof. The container can be labeled for use.

The kit can include informational material for performing andinterpreting the assay. The kit can also provide guidance as to where toreport the results of the assay, e.g., to a treatment center orhealthcare provider. The kit can include forms for reporting the resultsof a gene activity assay described herein, and address and contactinformation regarding where to send such forms or other relatedinformation; or a URL (Uniform Resource Locator) address for reportingthe results in an online database or an online application (e.g., anapp). In another embodiment, the informational material can includeguidance regarding whether a patient should receive treatment with ananti-cancer stem cell agent, depending on the results of the assay.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. The informational material of thekit can be contact information, e.g., a physical address, email address,website, or telephone number, where a user of the kit can obtainsubstantive information about the gene activity assay and/or its use inthe methods described herein. The informational material can also beprovided in any combination of formats.

A subject sample can be provided to an assay provider, e.g., a serviceprovider (such as a third party facility) or a healthcare provider thatevaluates the sample in an assay and provides a read out. For example,an assay provider can receive a sample from a subject, such as a tissuesample, or a plasma, blood or serum sample, and evaluate the sampleusing an assay described herein, and determines that the subject is acandidate to receive treatment with an inhibitor as described herein.The assay provider can inform a healthcare provider that the subject isa candidate for treatment with an inhibitor as described herein, and thecandidate is administered the inhibitor as described herein. The assayprovider can provide the results of the evaluation, and optionally,conclusions regarding one or more of diagnosis, prognosis, orappropriate therapy options to, for example, a healthcare provider, orpatient, or an insurance company, in any suitable format, such as bymail or electronically, or through an online database. The informationcollected and provided by the assay provider can be stored in adatabase.

EXAMPLES Example A

In this example, the enzymatic activity of glutaminase is measuredthrough a coupled endpoint assay. Glutamine and phosphate are suppliedto GAC at a concentration equal to Km and AC₅₀, respectively, and GACconcentration is adjusted to give a linear reaction for 60 minutes. Theglutamate produced is converted to 2-OG by a kinetic excess of glutamatedehydrogenase. This second step is configured for 2×Km for NAD, sinceexcess NAD is inhibitory. However, a kinetic excess of the thirdcoupling enzyme, diaphorase, recycles NAD from NADH to keep the NADconcentration constant during the timecourse of the assay. Diaphorase,also supplied in kinetic excess, oxidizes NADH produced by GDH back toNAD with the concomitant reduction of rezasurin to the highlyfluorescent resorufin. Resorufin in measured after the assay is stoppedwith SDS by Ex544/Em590. A reduction in the signal indicates inhibitionof some component of the coupled enzyme system. Prospective hits arecounterscreened against GDH/diaphorase alone to remove hits to thecoupling enzyme system in a second assay.

1. Materials

BSA Sigma #3294 (protease-free) diaphorase Worthington Enzyme LS004330.Resuspend at 10 mg/ml in ddH₂0 and store at −80 C. EDTA Sigma E6758 orequivalent glutamate dehydrogenase Sigma G7882 glutamine Sigma G3126 orequivalent HEPES (pH8.5) Sigma H3375 or equivalent, to pH 8.5 with NaOHNaCl Sigma S7653 or equivalent NAD Sigma N7004; note: power willdecompose to inhibitor if stored outside dessicator. Purchase small lotsand prepare stocks in solution and store at −80 C. resazurin Sigma199303 sodium dodecyl sulfate Sigma L4390 or equivalent sodium phosphatePrepare from Sigma monobasic (S8282) and (pH 8.5) dibasic (S7907)solutions or equivalents; 1M stock final concentration prepared from 1Mstocks of each of the dibasic and monobasic solutions.

2. Buffers

2× Buffer (300 mM NaCl, 100 mM HEPES pH 8.5, 0.1% BSA, 0.5 mM EDTA, 100mM sodium phosphate pH 8.5)5× Substrate Mix (1× Buffer final concentration, with 13 mM glutamine,100 μM resazurin, 50 μg/ml diaphorase)1.2× Enzyme Mix (1× Buffer final concentration, with 0.875 μg/ml GAC,1.56 mM NAD, 6.25 units/ml GDH)Stop Mix (6% SDS in ddH₂O)

Reaction Procedure

1. Add 1 μl compound in 100% DMSO2. Add 40 μl of Enzyme Mix and incubate for 60 minute at roomtemperature3. Add 10 μl of Substrate Mix to start reaction4. Stop reaction with 25 μl of 6% SDS and read Ex544 Em 590

Example B

In this example, the potential for a compound to inhibit the coupledenzyme assay system of the glutaminase HTS method, which comprisesglutamate dehydrogenase and diaphorase, is tested through a coupledendpoint assay. Glutamate is supplied at Km to GDH, which then performsa reductive deamidation to produce 2OG. NAD is supplied at 2×Km to thesystem, and its conversion to NADH is monitored by the activity ofdiaphorase. Diaphorase, supplied in large kinetic excess to GDH,converts NADH back to NAD to keep NAD levels constant in the reactionwhile at the same time reducing rezasurin to the highly fluorescentresorufin. Resorufin in measured after the assay is stopped with SDS byEx544/Em590. A reduction in the signal indicates inhibition of somecomponent of the coupled enzyme system.

3. Materials

BSA Sigma #3294 (protease-free) diaphorase Worthington Enzyme LS004330.Resuspend at 10 mg/ml in ddH₂0 and store at −80 C. EDTA Sigma E6758 orequivalent glutamate dehydrogenase Sigma G7882 glutamic acid Sigma G1251or equivalent HEPES (pH 8.5) Sigma H3375 or equivalent, to pH 8.5 withNaOH NaCl Sigma S7653 or equivalent NAD Sigma N7004; note: powder willdecompose to inhibitor if stored outside dessicator. Purchase small lotsand prepare stocks in solution and store at −80 C. resazurin Sigma199303 sodium dodecyl sulfate Sigma L4390 or equivalent

4. Buffers

2× Buffer (300 mM NaCl, 100 mM HEPES pH 8.5, 0.1% BSA, 0.5 mM EDTA, 100mM phosphate pH 8.5)2× Substrate Mix (1× Buffer final concentration, 40 μM resazurin, 1.8 mMglutamate, 20 μg/ml diaphorase)10×NAD Mix (1× Buffer final concentration, 12.5 mM NAD)2.5× Enzyme Mix (1× Buffer final concentration, GDH enzyme as determinedfor appropriate linearity; for example 0.05 units/ml as described hereto get 0.02 units/ml final concentration)

Reaction Procedure

1. Add 1 μl compound in 100% DMSO2. Add 20 μl of Enzyme Mix and incubate for 60 minutes at roomtemperature

3. Add 5 μl of NAD Mix

4. Add 25 μl of Substrate Mix to start reaction5. Stop reaction with 25 μl of 6% SDS and read Ex544 Em 590

Example 1

trans-cyclopropane-1,2-diyldimethanol (I-2)

Compound I-2 was prepared by following a reported literature procedure(Org. Synth. 2008, 85, 15). To a suspension of LiAlH₄ (1.5 equivalents)in THF was added (1S,2S)-diethyl cyclopropane-1,2-dicarboxylate (1equivalent) slowly at 0° C. The reaction mixture was then warmed to roomtemperature and refluxed for 2 h. After cooling, the heterogeneousmixture was stirred at room temperature for 18 h. The reaction wasquenched by careful addition of saturated NH₄Cl solution followed byEtOAc. Stirring the reaction mass for next 5 h resulted in aprecipitation of a light yellow solid which was filtered through a padof celite. The celite layer was further washed with EtOAc. The combinedorganic layers were evaporated to obtain a pasty mass which was purifiedthrough column chromatography (80% EtOAC/hexane as eluent) to furnishthe title compound I-2.

trans-1,2-bis(bromomethyl)cyclopropane (I-3)

Compound I-3 was prepared by following a reported procedure (TetrahedronLett. 1997, 53, 10459). To a solution of triphenylphosphine (2.1equivalents) in DCM was added bromine (2.1 equivalents) slowly at 0° C.The reaction mixture was stirred for 0.25 h before addingtrans-cyclopropane-1,2-diyldimethanol I-2 (1 equivalent) (as a solutionin THF). It was then warmed to room temperature and stirred for an hour.In workup, all the volatiles were evaporated and the crude mass waspurified using column chromatography (30% EtOAc/hexane) to afford thetitle compound I-3.

trans-2,2′-(cyclopropane-1,2-diyl)diacetonitrile (I-4)

Compound I-4 was prepared by following a reported procedure (TetrahedronLett. 1997, 53, 10459). trans-1,2-bis(bromomethyl)cyclopropane 1-3 (1equivalent) was dissolved in a mixture of EtOH/water (2/1). Followingthe addition of NaCN (4 equivalents) the reaction mixture was refluxedovernight. All the volatile materials were evaporated to obtain a pastymass which was dissolved in water and extracted with ether. The aqueouslayer was further extracted with ether and the combined organic layerswere evaporated to obtain the title compound I-4. This material was usedfor the next step without any purification.

trans-5,5′-(-cyclopropane-1,2-diylbis(methylene))bis(1,3,4-thiadiazol-2-amine)(I-5)

trans-2,2′-(cyclopropane-1,2-diyl)diacetonitrile I-4 (1 equivalent) wasdissolved in TFA (2.0 mL) and thiosemicarbazide (2 equivalents) wasadded to it. The reaction mixture was refluxed for 3 h at 100° C. beforequenching by careful addition of a saturated solution of NaHCO₃ at 0° C.to bring the pH around 8-9. The solid precipitated was filtered andfurther purified to obtaintrans-5,5′-(-cyclopropane-1,2-diylbis(methylene))bis(1,3,4thiadiazol2-amine)I-5.

General procedure for the synthesis of compounds 1, 3, 5, and 6

To a suspension oftrans-5,5′-(-cyclopropane-1,2-diylbis(methylene))bis(1,3,4-thiadiazol-2-amine(I-5) (1 equivalent) and the appropriate acid (3 equivalents) in DMF wasadded PYBOP (3 equivalents) and DIPEA (6 equivalents) and stirred forovernight. Water was then added to the reaction mixture and theresulting material was filtered and purified to obtain the desiredcompound.

N,N′-(5,5′-(trans-cyclopropane-1,2-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) (1)

¹H NMR (400 MHz, DMSO-d₆): 0.5-0.7 (m, 2H), 1.0-1.2 (m, 2H), 2.8-3.0 (m,4H), 3.7-3.9 (s, 4H), 7.2 (m, 10H), 12.60 (brs, 2H). Mass (M⁺+1):505.10.

N,N′-(5,5′-(trans-cyclopropane-1,2-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(3)

¹H NMR (400 MHz, DMSO-d₆) δ: 0.62 (m, 2H), 1.10 (m, 2H), 2.82-3.02 (m,4H), 4.0 (s, 4H), 7.30 (t, 2H), 7.40 (d, 2H), 7.80 (t, 2H), 8.50 (d,2H), 12.68 (brs, 2H); Mass (M⁺+1): 507.05.

N,N′-(5,5′-(trans-cyclopropane-1,2-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-3-yl)acetamide)(5)

¹H NMR (400 MHz, DMSO-d₆): 0.58-0.65 (m, 2H), 1.02-1.16 (m, 2H),2.80-3.13 (m, 4H), 3.84 (s, 4H), 7.34-7.41 (m, 2H), 7.72-7.80 (m, 2H),8.41-8.58 (m, 4H), 12.63 (brs, 2H). Mass (M⁺+1): 507.05.

N,N′-(5,5′-(trans-cyclopropane-1,2-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(thiophen-2-yl)acetamide)(6)

¹H NMR (400 MHz, DMSO-d₆): 0.58-0.64 (m, 2H), 1.05-1.19 (m, 2H),2.82-3.04 (m, 4H), 4.03 (s, 4H), 6.93-7.03 (m, 4H), 7.42 (s, 2H), 12.62(brs, 2H). Mass (M⁺+1): 516.90.

Example 2

(3-methylenecyclobutyl)methanol (I-11)

Compound I-10 was bought from a commercial source and the hydrolysis ofI-10 was carried out following a literature procedure (J. Am. Chem. Soc.1958, 80, 5507). To a solution of 3-methylenecyclobutanecarbonitrileI-10 (1 equivalent) in aqueous EtOH (50%) was added KOH (4 equivalents)and the homogeneous mixture was heated to reflux for 2 h. Upon cooling,all the volatile materials were evaporated and the solid was suspendedin water. The pH of the solution was adjusted to 2 by the addition of 1NHCl and the desired compound was extracted with ethyl acetate. Thecombined organic layers were washed with brine, dried over anhydrousMgSO₄ and evaporated to obtain 3-methylenecyclobutanecarboxylic acid inquantitative yield. This material was carried forward directly for thenext step.

To a suspension of LiAlH₄ (1.5 equivalents) in THF was added3-methylenecyclobutanecarboxylic acid (in THF) (1 equivalent) slowly at0° C. The cooling bath was removed and the reaction mixture was warmedto room temperature and stirred for 3 h. Following a Fischer workup, thedesired compound I-11 was obtained. This material was used for the nextstep without any purification.

Cyclobutane-1,3-diyldimethanol (I-12)

(3-Methylenecyclobutyl)methanol I-11 (1 equivalent) was dissolved inanhydrous THF and BH₃.DMS (1 equivalent) was added drop wise at 0° C.Following the addition, the reaction mixture was warmed to roomtemperature and stirred overnight. The reaction was quenched by carefuladdition of NaOH (3M solution in water) at O° C. followed by theaddition of H₂O₂. Stirring was continued for 3 h at room temperaturebefore diluting with water and extracting the diol with ethyl acetate.The crude material was taken directly for the next step without anypurification.

Cyclobutane-1,3-diylbis(methylene)bis(4-methylbenzenesulfonate) (I-13)

A solution of cyclobutane-1,3-diyldimethanol I-12 (1 equivalent) and TEA(3 equivalents) in dichloromethane was cooled to 0° C. and tosylchloride(2 equivalents) was added in portion. The reaction mixture was left for12 h. During workup, the reaction mixture was diluted withdichloromethane and the organic layer was washed with water. The organiclayer was concentrated to afford the desired product I-13.

2,2′-(Cyclobutane-1,3-diyl)diacetonitrile (I-14)

To a solution ofcyclobutane-1,3-diylbis(methylene)bis(4-methylbenzenesulfonate) I-13 (1equivalent) in DMF was added NaCN (6 equivalents) and was refluxed for12 h. During workup, the reaction mixture was diluted with water and thedesired compound was extracted with ethyl acetate. Evaporation of theorganic solvent furnished I-14.

5,5′-(cyclobutane-1,3-diylbis(methylene))bis(1,3,4-thiadiazol-2-amine)(I-15)

To a solution of 2,2′-(Cyclobutane-1,3-diyl)diacetonitrile I-14 (1equivalent) in TFA was added thiosemicarbazide (2 equivalents) and thesolution was stirred at 100° C. for 3 h. The reaction was cooled to roomtemperature and quenched with saturated NaHCO₃ solution. The materialwas filtered and washed with water, ethyl acetate and diethyl ethersequentially. The diamine I-15 was obtained.

General Procedure for the Synthesis of Compounds 7 and 8:

To a solution of the corresponding acid (2 equivalents) and DIPEA (6equivalents) in DMF (NMP for pyridyl derivative) was added PYBOP (3equivalents) at 0° C. and was stirred for 10 min at room temperature.Compound I-15 was added to the reaction mixture and stirring wascontinued overnight. Water was then added and the desired product wasextracted with ethyl acetate. Organic layer was dried over anhydroussodium sulfate and evaporated under reduced pressure. The crude productobtained was purified by standard methods to obtain the pure products.

N,N′-(5,5′-(cyclobutane-1,3-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(8)

¹H NMR (400 MHz, DMSO-d₆) δ: 0.9-1.2 (m, 1H), 1.42-1.60 (m, 1H),1.84-1.95 (m, 2H), 2.10-2.22 (m, 1H), 2.6-2.7 (m, 1H), 3.0-3.16 (m, 4H),3.80 (s, 4H), 7.10-7.40 (m, 10H), 12.62 (brs, 2H); Mass (M⁺+1): 519.19,541.25 (M⁺+23).

N,N′-(5,5′-(cyclobutane-1,3-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(7)

¹H NMR (400 MHz, DMSO-d₆) δ: 1.19-1.22 (m, 1H), 1.45-1.61 (m, 1H),1.82-1.92 (m, 1H), 2.15-2.05 (m, 1H), 3.01-3.16 (m, 4H), 4.01 (s, 4H),7.22-7.42 (m, 4H), 7.72-7.80 (m, 2H), 8.44 (s, 2H), 12.6 (brs, 2H). Mass(M⁺+23): 543.15.

Example 3

Cyclopentane-1,3-dicarbaldehyde (I-19)

KMnO₄ (3.2 equivalents) and CuSO₄.5H₂O (2 equivalents) were taken indichloromethane and distilled water was added to this suspension.Norbornene I-18 (1 equivalent) was dissolved in dichloromethane andadded slowly to the oxidant mixture followed by t-butanol. After 3 h thereaction mixture was filtered through Celite and washed with saturatedbrine. The brine was then re-washed with dichloromethane. The combinedorganic layers were dried over sodium sulfate, and the solvent wasremoved by rotary evaporation under ambient temperature to afford thedesired product I-19.

Cyclopentane-1,3-diyldimethanol (I-20)

To 1,3-cyclopentanedicarbaldehyde I-19 (1 equivalent) taken in a flaskwas added deoxygenated methanol. The reaction mixture was cooled to 0°C. and NaBH₄ (2 equivalents) was added in small aliquots in order toavoid a raise in the reaction temperature above 10° C. The reactionmixture was left to warm to room temperature and stirred for anadditional 3 h under nitrogen atmosphere. Distilled water was added tothe reaction mixture to quench any remaining NaBH₄ and then rotaryevaporated to remove methanol from the solution. The remaining mixturewas extracted with dichloromethane (5 times) and the combined organicfractions were dried over anhydrous sodium sulfate and evaporated todryness to afford the title compound I-20.

Cyclopentane-1,3-diylbis(methylene)bis(4-methylbenzenesulfonate) (I-21)

Cyclopentane-1,3-diyldimethanol I-20 (1 equivalent) taken in pyridinewas cooled to 0° C. and p-toluene sulfonyl chloride (3 equivalents) wasadded portion wise and stirred at RT overnight. The progress of thereaction was monitored by TLC. After completion of the reaction,pyridine was distilled off. Residue was diluted with diethyl ether,washed with 1N HCl, NaHCO₃ solution, brine, dried over anhydrous sodiumsulfate and evaporated under reduced pressure. The crude product waspurified by a standard method to afford the desired product I-21.

2,2′-(cyclopentane-1,3-diyl)diacetonitrile (I-22)

Cyclopentane-1,3-diylbis(methylene)bis(4-methylbenzenesulfonate) 1-21 (1equivalent) was taken in DMF:H₂O mixture (3:1) and sodium cyanide (6equivalents) was added and stirred at 150° C. for 15 h. The progress ofthe reaction was monitored by TLC. After completion of the reaction,reaction mixture was quenched with water, extracted with diethyl ether.The organic layer was washed with brine, dried over anhydrous sodiumsulfate and evaporated under reduced pressure. The crude product waspurified to afford the desired product I-22.

5,5′-(cyclopentane-1,3-diylbis(methylene))bis(1,3,4-thiadiazol-2-amine)(I-23)

The title compound was synthesized from2,2′-(cyclopentane-1,3-diyl)diacetonitrile 1-22 by following theprocedure described for compounds I-15.

N,N′-(5,5′-(cyclopentane-1,3-diylbis(methylene))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(4)

The title compound was synthesized from5,5′-(cyclopentane-1,3-diylbis-(methylene))bis(1,3,4thiadiazol-2-amine)I-15 by following the general procedure as described for compound 7 and8.

¹H NMR (400 MHz, DMSO-d₆) δ: 1.35-1.40 (m, 2H), 1.70-1.80 (m, 2H),1.90-1.95 (m, 1H), 2.20-2.24 (m, 2H), 2.98 (d, 4H), 4.00 (s, 4H), 7.26(t, 2H), 7.39 (d, 2H), 7.78 (t, 2H), 8.50 (d, 2H), 12.70 (brs, 2H); Mass(M⁺+1): 535.10.

Example 4

N,N′-(5,5′-((1R,3R)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(19)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.92-1.95 (m, 2H), 2.26-2.29 (m, 2H),2.34-2.37 (t, 2H), 3.70 (s, 2H), 3.72-3.76 (m, 4H), 7.24-7.34 (m, 10H),11.8 (s, 2H); Mass (M⁺+1): 505.7.

N,N′-(5,5′-((1R,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(18)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.94-1.97 (m, 2H), 2.08-2.09 (t, 1H),2.19-2.23 (m, 2H), 2.61-2.64 (t, 1H), 3.63-3.66 (m, 2H), 3.75 (s, 4H),7.23-7.26 (m, 2H), 7.31-7.34 (t, 8H), 11.7 (s, 2H); Mass (M⁺+1): 505.7.

Example 5N,N′-(5,5′-((1R,4R)-cyclohexane-1,4-diylbis(methylene)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(2)

The title compound was synthesized from5,5′-((1R,4R)-cyclohexane1,4-diylbis(methylene))bis(1,3,4thiadiazol-2-amine)(trans) by following the general procedure as described for compound 7and 8.

¹H NMR (400 MHz, DMSO-d₆) δ: 1.0 (t, 2H), 1.60-1.80 (m, 8H), 2.85 (d,4H), 4.0 (s, 4H), 7.30 (t, 2H), 7.40 (d, 2H), 7.80 (t, 2H), 8.50 (d,2H), 12.65 (brs, 2H); Mass (M⁺+1) 549.10.

Example 6

5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (I-31)

A mixture of cyclohexane-1,3-dicarboxylic acid (1.0 equivalent) andthiosemicarbazide (2.0 equivalents) were taken in POCl₃ (6.0equivalents) and stirred at 80° C. for 3 h. The reaction mixture wascooled to room temperature and poured on to ice. The resulting mixturewas filtered and then brought to pH 8 using KOH. The resulting materialthat was formed washed with water and dried to afford the desiredproduct (31). This material was used as such for the next step.

General Procedure for the Synthesis of Compounds (I-33 to I-35):

A mixture of compound (I-31) (1 equivalent), the corresponding ester(2.5 equivalents), t-BuOK (3.0 equivalents) in DMF was stirred at120-140° C. for 30-60 min in a microwave oven. The resulting mixture waspurified by standard methods to afford the desired products.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1H-indol-3-yl)acetamide(22)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.58 (s, 2H), 1.60 (m, 4H), 2.27 (s, 2H),3.44 (s, 2H), 3.88 (s, 4H), 6.97-7.00 (t, 4H), 7.28-7.36 (m, 4H),7.56-7.57 (d, 2H), 10.96 (s, 2H); Mass (M⁺+H): 596.7.

N,N′-(5,5′-((1S,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1H-indol-3-yl)acetamide)(23)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.49-1.55 (s, 3H), 1.75-1.77 (m, 1H), 1.88(m, 1H), 2.05-2.07 (m, 2H), 2.28-2.41 (d, 1H), 3.23-3.27 (t, 2H), 3.88(s, 4H), 6.97-7.00 (t, 4H), 7.28-7.36 (m, 4H), 7.56-7.57 (d, 2H), 10.95(s, 2H), 12.53 (s, 2H); Mass (M⁺+H): 596.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)Obis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1H-indol-4-yl)acetamide)(24)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.01 (s, 2H), 1.57-1.60 (s, 4H), 2.2 (S,2H), 3.45 (S, 2H), 4.01 (s, 4H), 6.55 (s, 2H), 6.94-6.96 (d, 2H),7.02-7.05 (d, 2H), 7.28-7.33 (m, 4H), 11.11 (s, 2H); Mass (M⁺+H): 596.7.

N,N′-(5,5′-((1S,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1H-indol-4-yl)acetamide)(25)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.49-54 (m, 3H), 1.74-1.76 (s, 2H),1.87-1.90 (m, 3H), 2.37-2.45 (m, 2H), 3.99 (s, 4H), 6.54 (s, 2H),6.94-6.96 (d, 2H), 7.03-7.04 (d, 2H), 7.2-7.31 (m, 4H), 11.11 (s, 2H);Mass (M⁺+H): 597.3.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyrimidin-2-yl)acetamide)(27)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.61-1.63 (m, 2H), 1.83-1.92 (d, 4H), 2.29(s, 2H), 3.46 (s, 2H), 4.11 (s, 4H), 7.40-7.42 (t, 2H), 8.75-8.77 (d,4H), 12.02 (s, 2H); Mass (M⁺+H): 522.8.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-3-yl)acetamide)(28)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.59-1.61 (m, 2H), 1.81-1.94 (d, 4H), 2.26(s, 2H), 3.44 (s, 2H), 3.83 (s, 4H), 7.34-7.37 (m, 2H), 7.72-7.74 (d,2H), 8.46-8.51 (d, 4H), 12.75 (s, 2H); Mass (M⁺+H): 521.3.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-4-yl)acetamide)(29)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.59-1.62 (m, 2H), 1.82-1.93 (d, 4H), 2.27(s, 2H), 3.45 (s, 2H), 3.85 (s, 4H), 7.33-7.34 (d, 4H), 8.51-8.52 (d,4H), 12.49 (s, 2H); Mass (M⁺+H): 521.2.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-methylisoxazol-5-yl)acetamide)(30)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.61-1.64 (t, 2H), 1.83-1.97 (m, 4H), 2.21(s, 6H), 2.30-2.32 (s, 2H), 3.49-3.51 (t, 2H), 4.07 (s, 4H), 6.30 (s,2H), 12.82 (s, 2H); Mass (M⁺+H): 529.2.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(dimethylamino)phenyl)acetamide)(31)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.59 (s, 2H), 1.80-1.84 (m, 4H), 2.27-2.29(t, 2H), 2.85 (s, 12H), 3.45-3.47 (t, 2H), 3.63 (, 4H), 6.67-6.69 (d,2H), 7.13-7.14 (d, 2H), 12.55 (s, 2H); Mass (M⁺+H): 605.3.

N,N′-(5,5′-((1R,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(10)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.16-2.04 (m, 2H), 2.32-2.37 (d, 1H),3.15-3.33 (d, 2H), 3.66 (s, 4H), 7.21-7.30 (m, 10H); Mass (M⁺+1): 519.7.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(11)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.5-1.61 (t, 2H), 1.91-1.93 (t, 4H), 2.29(s, 2H), 3.46-3.47 (d, 2H), 3.80 (s, 4H), 7.25-7.35 (m, 10H), 12.7 (s,2H); Mass (M⁺+1): 519.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))diacetamide(12)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.61-1.63 (t, 2H), 1.84-1.97 (m, 4H), 2.17(s, 6H), 2.30-2.32 (d, 2H), 3.46-3.48 (t, 2H), 12.2 (s, 2H); Mass(M⁺+1): 367.7.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(14)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.60-1.63 (t, 2H), 1.83-1.85 (t, 2H),1.91-1.95 (m, 2H), 2.28-2.30 (t, 2H), 3.45-3.47 (t, 2H), 3.97 (s, 4H),7.26-7.29 (m, 2H), 7.39-7.40 (d, 2H), 7.74-7.78 (m, 2H), 8.48-8.49 (t,2H), 12.1 (s, 2H); Mass (M⁺+1): 521.7.

N,N′-(5,5′-((1R,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1-methyl-1H-indol-3-yl)acetamide)(15)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.49-1.57 (m, 3H), 1.74-1.79 (t, 1H),1.88-1.91 (d, 1H), 2.05-2.08 (t, 2H), 2.38-2.40 (d, 1H), 3.23-3.28 (t,2H), 3.75 (s, 6H), 3.87 (s, 4H), 12.6 (s, 2H); Mass (M⁺+1): 625.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1-methyl-1H-indol-3-yl)acetamide)(16)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.57-1.59 (t, 2H), 1.81-1.92 (m, 4H),2.26-2.27 (d, 2H), 3.44-3.46 (t, 2H), 3.76 (s, 6H), 3.88 (s, 4H),7.01-7.04 (t, 2H), 7.13-7.16 (t, 2H), 7.27 (s, 2H), 7.39-7.41 (d, 2H),7.57-7.59 (d, 2H), 12.6 (s, 2H); Mass (M⁺+1): 625.7.

N,N′-(5,5′-((1R,3R)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(21)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.60-1.62 (t, 2H), 1.82-1.85 (m, 2H),1.92-1.96 (m, 2H), 2.29-2.31 (t, 2H), 3.47-3.49 (t, 2H), 4.00 (s, 4H),7.27-7.29 (m, 2H), 7.38-7.40 (d, 2H), 7.75-7.78 (m, 2H), 8.48-8.49 (d,2H), 12.6 (t, 2H); Mass (M⁺+1): 521.7.

N,N′-(5,5′-((1R,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(20)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.52-1.62 (m, 3H), 1.78-1.80 (d, 1H),1.90-1.93 (d, 1H), 2.07-2.10 (d, 2H), 2.42-2.44 (d, 1H), 3.26-3.34 (m,2H), 4.00 (s, 4H), 7.27-7.30 (m, 2H), 7.39-7.40 (d, 2H), 7.75-7.79 (m,2H), 8.48-8.49 (d, 2H), 12.6 (s, 2H); Mass (M⁺+1): 521.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide)(17)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.61-1.62 (d, 2H), 1.80-1.85 (m, 2H),1.92-1.94 (t, 2H), 2.29-2.31 (t, 2H), 3.47-3.49 (t, 2H), 4.00 (s, 4H),7.27-7.29 (m, 2H), 7.38-7.40 (d, 2H), 7.75-7.78 (m, 2H), 8.48-8.49 (d,2H), 12.6 (t, 2H); Mass (M⁺+1): 521.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide)(9)

¹H NMR (500 MHz, DMSO-d₆) δ: 1.59 (s, 2H), 1.83 (s, 2H), 1.91 (s, 2H),2.28 (s, 2H), 3.46 (s, 2H), 3.79 (s, 4H), 7.26-7.32 (t, 10H); Mass(M⁺+1): 519.7.

Example 75,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine)

Step A: Trans-Cyclohexyl 1,3-Dicarboxylic Acid

Cis, trans-cyclohexyl 1,3-dicarboxylic acid (4.00 g, 23.23 mmol) wasdissolved in concentrated ammonium hydroxide at 0° C. CaCl₂ (3.09 g,27.88 mmol) in water (5 mL) was added at 0° C. The resulting materialwas filtered off, and the filtrate was acidified with concentrated HCl.The resulting material was collected by filtration and dried in vacuo togive trans-cyclohexyl 1,3-dicarboxylic acid. ¹H NMR (DMSO-d₆) δ: 1.44(m, 2H), 1.61 (m, 4H), 1.79 (m, 2H), 2.53 (m, 2H), 12.13 (brs, 2H).LC-MS: m/z 171.2 (M−H)⁻

Step B: (S,S)-dibenzyl cyclohexyl 1,3-dicarboxylate

To a mixture of trans-cyclohexyl 1,3-dicarboxylic acid (1.46 g, 8.5mmol), Cs₂CO₃ (8.28 g, 25.5 mmol) in DMF (20 mL) was added BnBr (4.36 g,25.5 mmol). The mixture was stirred at rt under nitrogen for 3 h. Theresidue was diluted with water and extracted with ethyl acetate. Thecombined organic solution was washed with water, dried over Na₂SO₄ andconcentrated in vacuo. The crude product was purified by flashchromatography to afford 3 g trans-dibenzyl cyclohexyl1,3-dicarboxylate. ¹H NMR (CHLOROFORM-d) δ: 7.51-7.30 (m, 10H), 5.15 (s,4H), 2.84-2.72 (m, 2H), 2.06 (t, J=5.9 Hz, 2H), 1.77 (m, 4H), 1.56 (p,J=6.0 Hz, 2H). LC-MS: m/z (M+H)=353.4

Chiral SFC separation: 3 g of trans-dibenzyl cyclohexyl1,3-dicarboxylate was separated by chiral SFC to afford 1.4 g(S,S)-dibenzyl cyclohexyl 1,3-dicarboxylate (93%).

Step C: (S,S)-cyclohexyl 1,3-dicarboxylic acid

To a solution of 1 g (S,S)-dibenzyl cyclohexyl 1,3-dicarboxylate in 10mL MeOH was added 10% Pd on carbon (0.1 g). The suspension was flushedwith hydrogen and stirred for 20 min. It was then filtered andconcentrated to give the desired compound.

¹H NMR (DMSO-d₆) δ: 1.44 (m, 2H), 1.61 (m, 4H), 1.79 (m, 2H), 2.53 (m,2H), 12.13 (brs, 2H). LC-MS: m/z (M−H)=171.2

Step D: 5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine)

A mixture of (S,S)-cyclohexyl 1,3-dicarboxylic acid (500 mg, 0.3 mmol)and thiosemicarbazide (550 mg, 0.6 mmol) were taken up in POCl₃ (10 mL)and stirred at 40° C. for 30 min, 60° C. for 30 min, and 80° C. for 2 h.The reaction mixture was cooled to room temperature and poured onto ice.The resulting mixture was then basified to pH=8 using NaOH and filtratedto give crude desired compound. ¹H NMR (DMSO-d₆) δ 7.05 (s, 4H), 3.25(m, 2H), 2.12 (t, 2H J=5.6 Hz, 2H), 1.84 (m, 2H), 1.70 (m, 2H). LC-MS:m/z (M+H)=283.3

Racemic-Trans-5,5′-(cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine)

was synthesized with a similar procedure.

Step E: (S,S)-cyclohexyl 1,3-dicarboxyl bis((2S)-Bornane-10,2-sultamide)

A solution of (S,S)-cyclohexyl 1,3-dicarboxylic acid (800 mg, 4.65 mmol)in SOCl₂ was stirred at 80° C. for 1.5 h. The extra SOCl₂ was removedunder reduced pressure and the residue was used to next step directly.To a solution of (2S)-Bornane-10,2-sultam (2 g, 9.3 mmol) in toluene (20mL) was added NaH (60% in oil, 465 mg, 11.63 mmol) in portions at 0° C.and the reaction mixture was stirred at this temperature for 30 minCyclohexane-1,3-dicarbonyl dichloride (obtained from above) in toluene(5 mL) was added dropwise and the reaction was stirred at roomtemperature overnight. The resulting mixture was diluted with EtOAc (50mL) and water (10 mL), separated and the aqueous layer was extractedwith EtOAc (50 mLx2). The combined organic layers were washed withbrine, dried over Na₂SO₄, filtered and purified by flash chromatographyto give the desired compound.

¹H NMR (CHLOROFORM-d) δ 3.93 (dd, J=7.6, 4.8 Hz, 2H), 3.66-3.59 (m, 2H),3.50 (d, J=13.7 Hz, 2H), 3.45 (d, J=13.8 Hz, 2H), 2.12 (dd, J=13.8, 7.7Hz, 2H), 1.82-2.03 (m, 12H), 1.57-1.70 (m, 4H), 1.39 (dt, J=16.8, 9.5Hz, 4H), 1.16 (s, 6H), 0.98 (s, 6H). LC-MS: m/z (M+H)=567.3.

The product was recrystallized in EtOAc to give a sample for singlecrystal x-ray diffraction, the result of which confirmed that theconfiguration of the starting material diacid was (S,S).

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))dibutyramide(Compound 37)

A solution of5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (20mg, 0.07 mmol), butyric acid (18.5 mg, 0.21 mmol), HATU (80.8 mg, 0.21mmol), and N-ethyl-N-isopropylpropan-2-amine (29.3 mg, 0.23 mmol) inN,N-dimethylformamide (5 ml) was stirred at room temperature overnight.The mixture was poured into water (10 ml), the precipitate was filteredto give the crude product. The crude product was purified by a standardmethod to give the desired product.

¹H NMR (DMSO-d₆) δ: 12.41 (s, 2H), 3.43-3.55 (m, 2H), 2.44 (t, J=7.3 Hz,4H), 2.31 (t, J=5.7 Hz, 2H), 1.91-2.02 (m, 2H), 1.82-1.91 (m, 2H),1.57-1.68 (m, 6H), 0.90 (t, J=7.5 Hz, 6H). LC-MS: m/z (M+H)=423.6.

The following compounds were prepared in an analogous manner:

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(tetrahydrofuran-3-carboxamide)(Compound 48)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.58 (br. s., 2H), 3.87-3.98 (m, 2H), 3.65-3.83 (m,6H), 3.47-3.55 (m, 2H), 3.27-3.34 (m, 2H), 2.31 (t, J=6.0 Hz, 2H),2.02-2.18 (m, 4H), 1.92-2.01 (m, 2H), 1.82-1.90 (m, 2H), 1.60-1.68 (m,2H). LC-MS: m/z (M+H)=479.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(4,4,4-trifluorobutanamide)(Compound 47)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.61 (br. s., 2H), 3.45-3.55 (m, 2H), 2.73-2.82 (q,4H), 2.56-2.72 (m, 4H), 2.32 (t, J=5.6 Hz, 2H), 1.92-2.01 (m, 2H),1.81-1.90 (m, 2H), 1.57-1.70 (m, 2H). LC-MS: m/z (M+H)=531.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-cyclopropylacetamide)(Compound 49)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.39 (s, 2H), 3.44-3.55 (m, 2H), 2.28-2.41 (m, 6H),1.92-2.02 (m, 2H), 1.82-1.92 (m, 2H), 1.60-1.70 (m, 2H), 0.99-1.11 (m,2H), 0.46-0.54 (m, 4H), 0.17-0.25 (m, 4H). LC-MS: m/z (M+H)=447.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(methylthio)acetamide)(Compound 60)

The procedure was the same as Compound 37.

¹H NMR (DMSO-d₆) δ: 12.58 (br. s., 2H), 3.47-3.54 (m, 2H), 3.41 (s, 4H),2.33 (t, J=5.7 Hz, 2H), 2.16 (s, 6H), 1.85-1.99 (m, 4H), 1.61-1.67 (m,2H). LC-MS: m/z (M+H)=459.5

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(3-(methylthio)propanamide)(Compound 46)

The procedure was the same as Compound 37.

¹H NMR (DMSO-d₆) δ: 12.51 (br. s., 2H), 3.50 (m, 2H), 2.77 (br. s, 8H),2.32 (m, 2H), 2.08 (s, 6H), 1.96 (m, 2H), 1.87 (m, 2H), 1.64 (m, 2H).LC-MS: m/z (M+H)=487.5

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(tetrahydrofuran-2-yl)acetamide)(Compound 77)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 11.96 (br. s., 2H), 4.35 (quin, J=6.6 Hz, 2H),3.93-4.00 (m, 2H), 3.80-3.86 (m, 2H), 3.55-3.62 (m, 2H), 2.85 (d, J=6.2Hz, 4H), 2.48 (t, J=5.5 Hz, 2H), 2.12-2.18 (m, 2H), 1.95-2.00 (m, 4H),1.76-1.82 (m, 4H), 1.61-1.68 (m, 4H). LC-MS: m/z (M+H)=507.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-oxopyrrolidin-1-yl)acetamide)(Compound 85)

The procedure was the same as Compound 37

1H NMR (CHLOROFORM-d) δ: 4.48 (s, 4H), 3.53-3.61 (m, 6H), 2.49 (t, J=7.9Hz, 4H), 2.43 (t, J=5.4 Hz, 2H), 2.08-2.16 (m, 4H), 2.03 (m, 2H), 1.93(m, 2H), 1.67-1.77 (m, 2H). LC-MS: m/z (M+H)=533.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))dibenzamide(Compound 39)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.98 (br. s., 2H), 8.12 (d, J=7.3 Hz, 4H),7.63-7.72 (m, 2H), 7.53-7.61 (m, 4H), 3.57 (m, 2H), 2.37-2.45 (t, J=5.6Hz, 2H), 1.98 (m, 2H), 1.89 (m, 2H), 1.70 (m, 2H). LC-MS: m/z(M+H)=491.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))diacetamide(Compound 41)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.44 (s, 2H), 3.48 (m, 2H), 2.27-2.35 (t, J=5.6 Hz,2H), 2.18 (s, 6H), 1.95 (m, 2H), 1.87 (m, 2H), 1.59-1.67 (m, 2H). LC-MS:m/z (M+H)=367.5

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))dipropionamide(Compound 40)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.40 (s, 2H), 3.37-3.54 (m, 2H), 2.47 (q, J=7.6 Hz,4H), 2.31 (t, J=5.6 Hz, 2H), 1.91-2.06 (m, 2H), 1.75-1.91 (m, 2H),1.52-1.75 (m, 2H), 1.09 (t, J=7.5 Hz, 6H). LC-MS: m/z (M+H)=395.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-methylpropanamide)((Compound 98)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.42 (s, 2H), 3.43-3.53 (m, 2H), 2.77 (septet,J=6.7 Hz, 2H), 2.31 (t, J=5.8 Hz, 2H), 1.91-2.02 (m, 2H), 1.80-1.91 (m,2H), 1.57-1.68 (m, 2H), 1.12 (d, J=7.6 Hz, 12H). LC-MS: m/z (M+H)=423.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))dicyclopentanecarboxamide(Compound 62)

¹H NMR (DMSO-d₆) δ 3.48 (m, 2H), 2.95 (m, 2H), 2.31 (t, J=5.8 Hz, 2H),2.04-1.85 (m, 8H), 1.76-1.60 (m, 8H), 1.57 (m, 2H). LC-MS: m/z(M+H)=475.2

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(3-hydroxy-2,2-dimethylpropanamide)(Compound 63)

¹H NMR (DMSO-d₆) δ 3.82 (s, 4H), 3.57 (m, 2H), 2.46 (t, J=5.8 Hz, 2H),2.05 (m, 2H), 1.98 (m, 2H), 1.73 (m, 2H), 1.29 (s, 12H). LC-MS: m/z(M+H)=483.2.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-fluoro-3-methoxyphenyl)acetamide)(Compound 78)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 7.13-7.18 (m, 4H), 6.88 (d, J=2.4 Hz, 2H), 3.83 (s,6H), 3.78 (s, 4H), 2.29 (t, J=5.4 Hz, 2H), 1.93 (m, 2H), 1.84 (m, 2H),1.60 (m, 2H). LC-MS: m/z (M+H)=615.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(benzo[d][1,3]dioxol-5-yl)acetamide)(Compound 86)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 6.98 (s, 2H), 6.89-6.94 (m, J=7.8 Hz, 2H),6.69-6.73 (m, J=8.0 Hz, 2H), 5.89 (s, 4H), 3.93 (s, 4H), 3.56-3.64 (m,2H), 2.49 (t, J=5.4 Hz, 2H), 2.02-2.10 (m, 2H), 1.99 (m, 2H), 1.76-1.82(m, 2H). LC-MS: m/z (M+H)=607.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-fluorophenyl)acetamide)(Compound 88)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.70 (s, 2H), 7.36 (dd, J=8.6, 5.6 Hz, 4H), 7.16(t, J=8.9 Hz, 4H), 3.81 (s, 4H), 3.43-3.51 (m, 2H), 2.29 (t, J=5.6 Hz,2H), 1.89-1.97 (m, 2H), 1.85 (m, 2H), 1.57-1.64 (m, 2H). LC-MS: m/z(M+H)=555.7

N,N′-(5,5′-(1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2,6-difluorophenyl)acetamide)(Compound 86)

The procedure was the same as Compound 37.

¹H NMR (DMSO-d₆) δ: 12.83 (br. s., 2H), 7.42 (m, 2H), 7.12 (t, J=7.9 Hz,4H), 3.93 (s, 4H), 3.47-3.54 (m, 2H), 2.31 (t, J=5.6 Hz, 2H), 1.89-1.99(m, 2H), 1.80-1.89 (m, 2H), 1.58-1.67 (m, 2H). LC-MS: m/z (M+H)=591.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-methoxyphenyl)acetamide)(Compound 42)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.59 (s, 2H), 7.24-7.30 (m, 2H), 7.21 (dd, J=7.5,1.6 Hz, 2H), 6.98 (d, J=7.9 Hz, 2H), 6.88-6.93 (m, 2H), 3.78 (s, 4H),3.74 (s, 6H), 3.49 (m, 2H), 2.30 (t, J=5.7 Hz, 2H), 1.91-1.99 (m, 2H),1.79-1.87 (m, 2H), 1.59-1.65 (m, 2H). LC-MS: m/z (M+H)=579.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-chlorophenyl)acetamide)(Compound 43)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.73 (s, 2H), 7.42 (s, 2H), 7.34-7.38 (m, 4H),7.27-7.31 (m, 2H), 3.84 (s, 4H), 3.43-3.52 (m, 2H), 2.30 (t, J=5.6 Hz,2H), 1.89-1.98 (m, 2H), 1.77-1.88 (m, 2H), 1.57-1.65 (m, 2H). LC-MS: m/z(M+H)=587.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-methoxyphenyl)acetamide)(Compound 44)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.69 (s, 2H), 7.24 (t, J=7.9 Hz, 2H), 6.91 (d,J=2.1 Hz, 2H), 6.89 (d, J=7.6 Hz, 2H), 6.84 (dd, J=8.2, 2.1 Hz, 2H),3.77 (s, 4H), 3.74 (s, 6H), 3.47 (dt, J=11.2, 5.7 Hz, 2H), 2.29 (t,J=5.7 Hz, 2H), 1.88-1.98 (m, 2H), 1.76-1.86 (m, 2H), 1.54-1.65 (m, 2H).LC-MS: m/z (M+H)=579.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-methoxyphenyl)acetamide)(Compound 52)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.67 (s, 2H), 7.24 (d, J=8.8 Hz, 4H), 6.89 (d,J=8.5 Hz, 4H), 3.73 (s, 6H), 3.72 (s, 4H), 3.43-3.50 (m, 2H), 2.29 (t,J=5.9 Hz, 2H), 1.89-2.00 (m, 2H), 1.78-1.87 (m, 2H), 1.55-1.66 (m, 2H).LC-MS: m/z (M+H)=579.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-chlorophenyl)acetamide)(Compound 51)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.77 (s, 2H), 7.41-7.48 (m, 4H), 7.29-7.37 (m, 4H),4.00 (s, 4H), 3.45-3.54 (m, 2H), 2.30 (t, J=5.4 Hz, 2H), 1.91-1.99 (m,2H), 1.78-1.88 (m, 2H), 1.56-1.68 (m, 2H). LC-MS: m/z (M+H)=587.6.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-chlorophenyl)acetamide)(Compound 50)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.73 (s, 2H), 7.40 (d, J=8.8 Hz, 4H), 7.35 (d,J=8.8 Hz, 4H), 3.82 (s, 4H), 3.42-3.51 (m, 2H), 2.29 (t, J=5.6 Hz, 2H),1.89-1.99 (m, 2H), 1.77-1.87 (m, 2H), 1.55-1.66 (m, 2H). LC-MS: m/z(M+H)=587.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(benzo[d][1,3]dioxol-4-yl)acetamide)(Compound 218)

The procedure was the same as Compound 37

¹HNMR (CHLOROFORM-d) δ: 6.78-6.84 (m, 6H), 5.99 (s, 4H), 3.98 (s, 4H),3.55-3.61 (m, 2H), 2.45 (t, J=5.4 Hz, 2H), 2.02 (m, 2H), 1.92 (m, 2H),1.74 (m, 2H). LC-MS: m/z (M+H)=607.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(methylsulfonyl)phenyl)acetamide)(Compound 54)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.78 (s, 2H), 7.92 (s, 2H), 7.83-7.88 (d, J=7.6 Hz,2H), 7.57-7.73 (m, 4H), 3.97 (s, 4H), 3.43-3.51 (m, 2H), 3.22 (s, 6H),2.30 (t, J=5.5 Hz, 2H), 1.90-1.98 (m, 2H), 1.76-1.88 (m, 2H), 1.55-1.65(m, 2H). LC-MS: m/z (M+H)=675.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(methylsulfonyl)phenyl)acetamide)(Compound 66)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.80 (s, 1H), 7.90 (d, J=8.3 Hz, 4H), 7.60 (d,J=8.3 Hz, 4H), 3.96 (s, 4H), 3.46 (m, 2H), 3.20 (s, 6H), 2.30 (t, J=5.5Hz, 2H), 1.92 (m, 2H), 1.83 (m, 2H), 1.61 (m, 2H). LC-MS: m/z(M+H)=675.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-methoxyphenyl)acetamide)(Compound 70)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.59 (s, 2H), 7.27 (t, J=7.7 Hz, 2H), 7.21 (d,J=7.5 Hz, 2H), 6.98 (d, J=8.3 Hz, 2H), 6.85-6.94 (m, 2H), 3.78 (s, 4H),3.74 (s, 6H), 3.49 (m, 2H), 2.30 (t, J=5.5 Hz, 2H), 1.90-1.98 (m, 2H),1.84 (m, 2H), 1.63 (m, 2H). LC-MS: m/z (M+H)=579.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-methoxyphenyl)acetamide)(Compound 71)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.68 (s, 2H), 7.25 (t, J=7.7 Hz, 2H), 6.8-6.91 (m,4H), 6.84 (d, J=7.5 Hz, 3H), 3.77 (s, 4H), 3.75 (s, 6H), 2.29 (t, J=5.4Hz, 2H), 1.91 (m, 2H), 1.84 (m, 2H), 1.61 (m, 2H). LC-MS: m/z(M+H)=579.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-cyanophenyl)acetamide)(Compound 72)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.86 (s, 2H), 7.85 (d, J=7.8 Hz, 2H), 7.70 (t,J=7.7 Hz, 2H), 7.56 (d, J=7.5 Hz, 2H), 7.46-7.54 (m, 2H), 4.11 (s, 4H),3.44-3.53 (m, 2H), 2.32 (t, J=5.1 Hz, 2H), 1.89-2.01 (m, 2H), 1.78-1.88(m, 2H), 1.58-1.67 (m, 2H). LC-MS: m/z (M+H)=569.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-cyanophenyl)acetamide)(Compound 73)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.75 (s, 2H), 7.79 (s, 2H), 7.76 (d, J=7.5 Hz, 2H),7.67 (d, J=7.8 Hz, 2H), 7.56 (t, J=7.8 Hz, 2H), 3.92 (s, 4H), 3.45-3.51(m, 2H), 2.30 (t, J=5.5 Hz, 2H), 1.90-1.99 (m, 2H), 1.80-1.88 (m, 2H),1.62 (m, 2H). LC-MS: m/z (M+H)=569.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-cyanophenyl)acetamide)(Compound 74)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.77 (s, 2H), 7.81 (d, J=8.3 Hz, 4H), 7.53 (d,J=8.3 Hz, 4H), 3.94 (s, 4H), 3.46 (m, 2H), 2.29 (t, J=5.5 Hz, 2H), 1.92(m, 2H), 1.83 (m, 2H), 1.61 (m, 2H). LC-MS: m/z (M+H)=569.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-(trifluoromethoxy)phenyl)acetamide)(Compound 75)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.77 (s, 2H), 7.50 (d, J=7.5 Hz, 2H), 7.41-7.47 (m,2H), 7.27-7.41 (m, 4H), 3.82-4.10 (s, 4H), 3.48-3.51 (m, 2H), 2.31 (t,J=5.4 Hz, 2H), 1.89-2.01 (m, 2H), 1.76-1.88 (m, 2H), 1.62 (m 2H). LC-MS:m/z (M+H)=687.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(trifluoromethoxy)phenyl)acetamide)(Compound 219)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 7.40-7.44 (d, J=7.8 Hz, 2H), 7.38 (s, 2H), 7.32(t, J=7.9 Hz, 2H), 7.09-7.12 (d, J=8.3 Hz, 2H), 4.08 (s, 4H), 3.61 (m,2H), 2.51 (t, J=5.4 Hz, 2H), 2.07 (m, 2H), 1.99 (m, 2H), 1.78 (m, 2H).LC-MS: m/z (M+H)=687.9.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-o-tolylacetamide)(Compound 83)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.70 (s, 2H), 7.22-7.26 (m, 2H), 7.16-7.20 (m, 4H),7.10-7.16 (m, 2H), 3.84 (s, 4H), 3.44-3.52 (m, 2H), 2.30 (t, J=5.4 Hz,2H), 2.27 (s, 6H), 1.89-2.01 (m, 2H), 1.76-1.88 (m, 2H), 1.56-1.66 (m,2H). LC-MS: m/z (M+H)=547.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-(trifluoromethyl)phenyl)acetamide)(Compound 82)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.75 (s, 2H), 7.72 (b.s., 2H), 7.55-7.67 (m, 6H),3.95 (s, 4H), 3.45-3.51 (m, 2H), 2.29 (t, J=5.6 Hz, 2H), 1.88-2.04 (m,2H), 1.74-1.88 (m, 2H), 1.62 (m, 2H). LC-MS: m/z (M+H)=655.8.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(dimethylamino)phenyl)acetamide)(Compound 35)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.66 (s, 2H), 7.12 (t, J=7.9 Hz, 2H), 6.71 (s, 2H),6.57-6.65 (m, 4H), 3.70 (s, 4H), 3.43-3.50 (m, 2H), 2.88 (s, 12H), 2.30(d, J=5.1 Hz, 2H), 1.88-1.97 (m, 2H), 1.77-1.88 (m, 2H), 1.56-1.63 (m,2H). LC-MS: m/z (M+H)=605.8.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-fluorophenyl)acetamide)(Compound 87)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.75 (s, 2H), 7.32-7.42 (m, 4H), 7.15-7.22 (m, 4H),3.90 (s, 4H), 3.44-3.54 (m, 2H), 2.30 (t, J=5.8 Hz, 2H), 1.89-1.98 (m,2H), 1.80-1.89 (m, 2H), 1.59-1.67 (m, 2H). LC-MS: m/z (M+H)=555.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-isopropoxyphenyl)acetamide)(Compound 92)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 10.35 (br. s., 2H), 7.26-7.28 (m, 4H),7.22-7.28 (m, 4H), 6.91-6.94 (m, 4H), 4.73 (septet, J=6.0 Hz, 2H), 3.85(s, 4H), 3.49-3.56 (m, 2H), 2.44 (t, J=4.8 Hz, 2H), 1.92-2.03 (m, 4H),1.72-1.76 (m, 2H), 1.43 (d, J=5.9 Hz, 12H). LC-MS: m/z (M+H)=635.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-isopropoxyphenyl)acetamide)(Compound 91)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 7.15-7.21 (m, 2H), 7.01-7.04 (m, 4H), 6.74-6.78(m, 2H), 4.45-4.53 (m, 2H), 3.99 (s, 4H), 3.57-3.63 (m, 2H), 2.52 (t,J=4.8 Hz, 2H), 2.08 (m, 2H), 2.00-2.05 (m, 2H), 1.81 (m, 2H), 1.28 (d,J=6.2 Hz, 12H). LC-MS: m/z (M+H)=635.9

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-bromophenyl)acetamide)(Compound 220)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 7.66 (s, 2H), 7.40 (d, J=7.8 Hz, 2H), 7.34-7.37(m, 2H), 7.13-7.17 (m, 2H), 3.98 (s, 4H), 3.62 (m, 2H), 2.54 (t, J=5.0Hz, 2H), 2.08 (m, 2H), 2.00-2.05 (m, 2H), 1.80 (m, 2H). LC-MS: m/z(M+H)=674.9

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-m-tolylacetamide)(Compound III)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.72 (s, 2H), 7.23-7.25 (m, 2H), 7.14-7.17 (m, 6H),3.84 (s, 4H), 3.45-3.50 (m, 2H), 2.30 (t, J=5.8 Hz, 2H), 2.26 (s, 6H),1.88-1.96 (m, 2H), 1.81-1.88 (m, 2H), 1.58-1.64 (m, 2H). LC-MS: m/z(M+H)=547.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2,3-dimethoxyphenyl)acetamide)(Compound 112)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 7.00-7.05 (m, 2H), 6.88 (d, J=8.1 Hz, 2H), 6.91(d, J=6.7 Hz, 2H), 3.99 (s, 4H), 3.86 (s, 12H), 3.48-3.55 (m, 2H), 2.41(t, J=5.2 Hz, 2H), 1.87-2.01 (m, 4H), 1.68-1.76 (m, 2H). LC-MS: m/z(M+H)=639.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-methoxypyridin-3-yl)acetamide)(Compound 221)

The procedure was the same as Compound 37.

¹H NMR (DMSO-d₆) δ: 8.21 (s, 2H), 7.70 (dd, J=8.6, 2.1 Hz, 2H), 6.70 (d,J=8.3 Hz, 2H), 3.97 (s, 4H), 3.88 (s, 6H), 3.56-3.62 (m, 2H), 2.50 (t,J=5.4 Hz, 2H), 2.06 (m, 2H), 1.98 (m, 2H), 1.78 (m, 2H). LC-MS: m/z(M+H)=581.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(thiophen-2-yl)acetamide)(Compound 59)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.74 (br. s., 2H), 7.43 (d., 2H), 7.01 (br. s.,4H), 4.05 (s, 4H), 3.49 (m, 2H), 2.31 (br. s., 2H), 1.94 (m, 2H), 1.86(m, 2H), 1.62 (m, 2H). LC-MS: m/z (M+H)=531.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(thiazol-4-yl)acetamide)(Compound 222)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 8.88 (d, J=1.9 Hz, 2H), 7.37-7.42 (d, J=1.9 Hz,2H), 4.22 (s, 4H), 3.55-3.61 (m, 2H), 2.47 (t, J=5.4 Hz, 2H), 2.03 (d,J=5.4 Hz, 2H), 1.93-1.98 (m, 2H), 1.71-1.77 (m, 2H). LC-MS: m/z(M+H)=533.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-methylisoxazol-5-yl)acetamide)(Compound 30)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 6.24 (s, 2H), 4.26 (s, 4H), 3.60 (s, 2H), 2.49(t, J=4.8 Hz, 2H), 2.28 (s, 6H), 2.07 (m, 2H), 1.99 (m, 2H), 1.78 (m,2H). LC-MS: m/z (M+H)=529.7.

Step A:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-nitrophenyl)acetamide)(Compound 57)

The procedure was analogous to Compound 37

¹H NMR (DMSO-d₆) δ: 12.78 (s, 2H), 8.11 (d, J=7.6 Hz, 2H), 7.73-7.78 (m,2H), 7.58-7.63 (m, 4H), 4.27 (s, 4H), 3.44-3.53 (m, 2H), 2.30 (t, J=5.7Hz, 2H), 1.88-1.98 (m, 2H), 1.76-1.88 (m, 2H), 1.55-1.67 (m, 2H). LC-MS:m/z (M+H)=609.7

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-aminophenyl)acetamide)(Compound 56)

The solution ofN,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-nitrophenyl)acetamide)(30 mg, 0.05 mmol) and Pd/C (3 mg) in methanol was stirred at roomtemperature for 3 h. The mixture was filtered and evaporated in vacuumto dryness. The residue was purified by a standard method to afforddesired compound.

¹H NMR (DMSO-d₆) δ: 12.58 (s, 2H), 7.01-7.07 (m, 2H), 6.94-7.00 (m, 2H),6.66 (d, J=7.0 Hz, 2H), 6.50-6.57 (m, 2H), 5.03 (b, 4H), 3.65 (s, 4H),3.45-3.50 (m, 2H), 2.30 (t, J=5.3 Hz, 2H), 1.90-2.00 (m, 2H), 1.79-1.88(m, 2H), 1.57-1.64 (m, 2H). LC-MS: m/z (M+H)=549.7

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-acetamidophenyl)acetamide)(Compound 55)

A solution of acetic acid (11.5 mg, 0.19 mmol), HATU (72.6 mg, 0.19mmol), and N-ethyl-N-isopropylpropan-2-amine (26.5 mg, 0.20 mmol) inN,N-dimethylformamide (2 ml) was stirred at room temperature for 15 min,then,N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-aminophenyl)acetamide)(35 mg, 0.06 mmol) was added and continued to stir overnight. Themixture was poured into water (10 ml), the precipitate was filtered togive the crude product. The crude product was purified by a standardmethod to give the desired product.

¹H NMR (DMSO-d₆) δ: 12.61 (s, 2H), 9.45 (s, 2H), 7.38 (d, J=7.6 Hz, 2H),7.23-7.32 (m, 4H), 7.11-7.20 (m, 2H), 3.83 (s, 4H), 3.47 (m, 2H), 2.30(t, J=5.6 Hz, 2H), 2.03 (s, 6H), 1.94 (m, 2H), 1.84 (m, 2H), 1.62 (m,2H). LC-MS: m/z (M+H)=633.7

Step A:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-nitrophenyl)acetamide)(Compound 69)

The procedure was analogous to Compound 37

¹H NMR (DMSO-d₆) δ: 12.79 (s, 2H), 8.25 (s, 2H), 8.16 (d, J=8.3 Hz, 2H),7.79 (d, J=7.5 Hz, 2H), 7.65 (t, J=7.9 Hz, 2H), 4.02 (s, 4H), 3.48 (m,2H), 2.30 (d, J=5.4 Hz, 2H), 1.89-2.00 (m, 2H), 1.83 (m, 2H), 1.62 (m,2H). LC-MS: m/z (M+H)=609.7

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-aminophenyl)acetamide)(Compound 53)

The procedure was the same as Compound 56

¹H NMR (DMSO-d₆) δ: 12.64 (s, 2H), 6.95 (t, J=7.8 Hz, 2H), 6.51 (s, 2H),6.40-6.47 (m, 4H), 5.07 (s, 4H), 3.61 (s, 4H), 3.48 (m, 2H), 2.30 (t,J=5.6 Hz, 2H), 1.92 (m, 2H), 1.75-1.88 (m, 2H), 1.62 (m, 2H). LC-MS: m/z(M+H)=549.7

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-acetamidophenyl)acetamide)(Compound 65)

The procedure was the same as Compound 55

¹H NMR (DMSO-d₆) δ: 12.73 (s, 2H), 9.94 (s, 2H), 7.54 (s, 2H), 7.50 (d,J=8.1 Hz, 2H), 7.24 (t, J=7.8 Hz, 2H), 6.94-7.03 (d, J=7.5 Hz, 2H), 3.76(s, 4H), 3.43-3.52 (m, 2H), 2.30 (t, J=5.6 Hz, 2H), 2.03 (s, 6H),1.90-1.98 (m, 2H), 1.77-1.89 (m, 2H), 1.57-1.65 (m, 2H). LC-MS: m/z(M+H)=633.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-nitrophenyl)acetamide)(Compound 64)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.81 (s, 2H), 8.20 (d, J=8.6 Hz, 4H), 7.61 (d,J=8.6 Hz, 4H), 4.01 (s, 4H), 3.40-3.55 (m, 2H), 2.29 (t, J=5.6 Hz, 2H),1.89-1.99 (m, 2H), 1.77-1.88 (m, 2H), 1.55-1.66 (m, 2H). LC-MS: m/z(M+H)=609.7.

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-acetamidophenyl)acetamide)(Compound 106)

The procedure was the same as Compound 55

¹H NMR (DMSO-d₆) δ: 12.67 (s, 2H), 9.93 (s, 2H), 7.52 (d, J=8.6 Hz, 4H),7.23 (d, J=8.3 Hz, 4H), 3.74 (s, 4H), 3.44-3.49 (m, 2H), 2.29 (t, J=5.5Hz, 2H), 2.03 (s, 6H), 1.90-1.97 (m, 2H), 1.76-1.86 (m, 2H), 1.51-1.65(m, 2H). LC-MS: m/z (M+H)=633.8.

Step A:(2E,2′E)-N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(3-(pyridin-2-yl)acrylamide)(Compound 45)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.88 (s, 2H), 8.69 (d, J=4.7 Hz, 2H), 7.90 (td,J=7.8, 1.8 Hz, 2H), 7.79 (d, J=15.4 Hz, 2H), 7.70 (d, J=7.8 Hz, 2H),7.44 (dd, J=7.1, 5.2 Hz, 2H), 7.38 (d, J=15.4 Hz, 2H), 3.52-3.59 (m,2H), 2.37 (t, J=5.4 Hz, 2H), 1.96-2.05 (m, 2H), 1.88-1.96 (m, 2H),1.64-1.71 (m, 2H); LC-MS: m/z (M+H)=545.7.

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(3-(pyridin-2-yl)propanamide)(Compound 80)

A solution of 50 mg of Compound 45 was dissolved in 2 mL MeOH. 5 mg Pd/Cwas added and the suspension was degassed and hydrogenated for 30 min.It was then filtered and concentrated to get the title compound 80.

¹H NMR (CHLOROFORM-d) δ: 8.59 (d, J=4.6 Hz, 2H), 7.64 (td, J=7.7, 1.4Hz, 2H), 7.27 (d, J=7.7 Hz 2H), 7.18 (dd, J=7.3, 5.1 Hz, 2H), 3.55 (d,J=5.6 Hz, 2H), 3.27-3.32 (m, 4H), 3.11-3.17 (m, 4H), 2.43 (t, J=5.4 Hz,2H), 1.94-2.03 (m, 4H), 1.74 (m, 2H). LC-MS: m/z (M+H)=549.7.

Step A: methyl 2-(6-methoxypyridin-2-yl)acetate

To the solution of LDA (18.3 mL, 36.5 mmol) in THF (120 mL) cooled to−78° C. was added 2-methoxy-6-methylpyridine (1.5 g, 12.2 mmol) in THF(15 mL) dropwise, and then the mixture was stirred at −78 degree for 2h. Dimethyl carbonate (1.2 mL, 14.6 mmol) was added quickly andcontinued to stir at −78° C. for 15 min. The reaction was quenched byH₂O at −78° C. The solution was extracted with ethyl acetate, dried oversodium sulfate and evaporated under reduced pressure. The residue waspurified with a standard method to give desired compound.

¹H NMR (CHLOROFORM-d) δ: 7.55 (dd, J=8.3, 7.3 Hz, 1H), 6.85 (d, J=7.3Hz, 1H), 6.65 (d, J=8.3 Hz, 1H), 3.92 (s, 3H), 3.77 (s, 2H), 3.74 (s,3H). LC-MS: m/z (M+H)=182.6.

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-methoxypyridin-2-yl)acetamide)(Compound 84)

A mixture of methyl 2-(6-methoxypyridin-2-yl)acetate (128.7 mg, 0.71mmol), 5,5-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine)(50 mg, 0.18 mmol), cesium carbonate (346.1 mg, 1.06 mmol) in DMF (3 mL)was heated to 130° C. under nitrogen atmosphere and microwave for 45min. The mixture was evaporated in vacuum to dryness. The residue waspurified by a standard method to afford desired compound.

¹H NMR (DMSO-d₆) δ: 12.70 (s, 2H), 7.60-7.76 (m, 2H), 6.98 (d, J=7.3 Hz,2H), 6.72 (d, J=8.1 Hz, 2H), 3.92 (s, 4H), 3.79 (s, 6H), 3.49 (m, J=6.2Hz, 2H), 2.32 (t, J=6.2 Hz, 2H), 1.94 (m, 2H), 1.85 (m, 2H), 1.63 (m,2H). LC-MS: m/z (M+H)=581.7.

The procedure was the same as Compound 84

Step A: methyl 2-(6-bromopyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.55 (t, J=7.7 Hz, 1H), 7.42 (d, J=7.8 Hz, 1H),7.30 (d, J=7.5 Hz, 1H), 3.86 (s, 2H), 3.75 (s, 3H). LC-MS: m/z(M+H)=230.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-bromopyridin-2-yl)acetamide)(Compound 118)

¹H NMR (DMSO-d₆) δ: 7.71-7.82 (m, 2H), 7.58 (s, 2H), 7.47 (d, J=7.5 Hz,2H), 4.03 (s, 4H), 3.50 (m, 2H), 2.32 (t, J=6.4 Hz, 2H), 1.84-1.99 (m,4H), 1.63 (m, 2H). LC-MS: m/z (M+H)=677.6.

Compound 223

The procedure was the same as Compound 84

Step A: Methyl 2-(4-methoxypyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ 8.38 (d, J=6.0 Hz, 1H), 6.83 (d, J=2.4 Hz, 1H),6.73 (dd, J=6.0 Hz, 2.4 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 2H), 3.73 (s,3H). LC-MS: m/z 182.3 (M+H)

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-methoxypyridin-2-yl)acetamide)(Compound 223)

¹H NMR (METHANOL-d₄) δ 8.50 (d, J=6.0 Hz, 2H), 6.83 (s, 2H), 6.81 (d,J=6.0 Hz, 2H), 3.99 (s, 4H), 3.88 (s, 6H), 3.57 (m, 2H), 2.47 (t, J=5.6Hz, 2H), 2.03-1.96 (m, 4H), 1.77 (t, J=5.6 Hz, 2H). LC-MS: m/z 581.9(M+H)

Compound 224

The procedure was the same as Compound 84

Step B:2-(2-methoxypyridin-4-yl)-N-[5-[(1S,3S)-3-[5-[[2-(2-methoxypyridin-4-yl)acetyl]amino]-1,3,4-thiadiazol-2-yl]cyclohexyl]-1,3,4-thiadiazol-2-yl]acetamide(Compound 224)

¹H NMR (CHLOROFORM-d) δ: 8.11 (d, J=5.1 Hz, 2H), 7.02 (d, J=4.6 Hz, 2H),6.91 (s, 2H), 4.01 (s, 4H), 3.89 (s, 6H), 3.58-3.67 (m, 2H), 2.52 (t,J=6.4 Hz, 2H), 2.08 (m, 2H), 1.99-2.04 (m, 2H), 1.76-1.84 (m, 2H).LC-MS: m/z (M+H)=581.4

N,N′-(5,5′-trans-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-methoxypyridin-2-yl)acetamide)(Compound 116)

The procedure was the same as Step B of Compound 84

¹H NMR (METHANOL-d4) δ: 8.08 (dd, J=4.8, 1.1 Hz, 2H), 7.46 (dd, J=8.3,1.1 Hz, 2H), 7.36 (dd, J=8.6, 4.8 Hz, 2H), 3.94 (s, 4H), 3.49-3.60 (m,2H), 2.43 (t, J=5.8 Hz, 2H), 2.01-2.11 (m, 2H), 1.89-1.98 (m, 2H),1.67-1.80 (m, 2H). LC-MS: m/z (M+H)=581.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-methoxypyridin-2-yl)acetamide)(Compound 225)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 8.27 (d, J=3.0 Hz, 2H), 7.24-7.40 (d, J=8.6 Hz,2H), 7.17 (dd, J=8.6, 3.0 Hz, 2H), 4.09 (s, 4H), 3.83 (s, 6H), 3.55 (m,2H), 2.48 (t, J=5.6 Hz, 2H), 1.85-2.09 (m, 4H), 1.62-1.78 (m, 2H).LC-MS: m/z (M+H)=581.7

Compound 226

The procedure was the same as Compound 84

Step A: methyl 2-(5-ethoxypyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 8.24 (m, 1H), 7.09-7.27 (m, 2H), 4.07 (q, J=7.0Hz, 2H), 3.80 (s, 2H), 3.72 (s, 3H), 1.44 (t, J=7.0 Hz, 4H). LC-MS: m/z(M+H)=196.3

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-ethoxypyridin-2-yl)acetamide)(Compound 226)

¹H NMR (CHLOROFORM-d) δ: 8.28 (d, J=2.7 Hz, 2H), 7.23-7.33 (d, J=5.1 Hz,2H), 7.09-7.22 (dd, J=2.7 Hz, 5.1 Hz, 2H), 3.96-4.19 (m, 8H), 3.51-3.61(m, 2H), 2.45 (t, J=5.4 Hz 0.2H), 1.90-2.07 (m, 4H), 1.74 (m, 2H),1.33-1.49 (t, J=7.0 Hz, 6H). LC-MS: m/z (M+H)=609.2

Compound 227

Step A: methyl 2-(5-((tert-butyldimethylsilyl)oxy)pyridin-2-yl)acetate

The procedure was the same as Step A of Compound 84

Step B: methyl 2-(5-hydroxypyridin-2-yl)acetate

To the stirred solution of methyl2-(5-((tert-butyldimethylsilyl)oxy)pyridin-2-yl)acetate (1 g, 3.5 mmol)in THF was added TBAF (1M, 3.5 ml) at room temperature. The wholemixture was stirred at room temperature overnight. It was evaporated andextracted with EtOAc, the organic layer was washed with water,evaporated and purified by a standard method.

¹H NMR (CHLOROFORM-d) δ: 8.18 (s, 1H), 7.23-7.32 (m, 2H), 3.84 (s, 2H),3.70 (s, 3H). LC-MS: m/z (M+H)=168.2

Step C: methyl 2-(5-(2-methoxyethoxy)pyridin-2-yl)acetate

To a stirred solution of methyl2-(5-((tert-butyldimethylsilyl)oxy)pyridin-2-yl)acetate (500 mg, 3 mmol)in MeCN was added Cs₂CO₃ (1.4 g, 4.5 mmol) followed by1-bromo-2-methoxyethane (621 mg, 4.5 mmol) at room temperature. Thewhole mixture was heated to 80° overnight. LC-MS showed desired product.It was filtered, evaporated and purified by a standard method.

¹H NMR (CHLOROFORM-d) δ: 8.28 (s, 1H), 7.22 (d, J=1.9 Hz, 2H), 4.16 (dd,J=5.5, 3.9 Hz, 2H), 3.80 (s, 2H), 3.74-3.78 (dd, J=5.5, 3.9 Hz, 2H),3.72 (s, 3H), 3.46 (s, 3H). LC-MS: m/z (M+H)=226.2

Step D:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-(2-methoxyethoxy)pyridin-2-yl)acetamide)(Compound 227)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 8.40 (d, J=2.4 Hz, 2H), 7.21-7.25 (dd, J=8.6,2.4 Hz, 2H), 7.19 (d, J=8.9 Hz, 2H), 4.17-4.23 (t, J=4.0 Hz, 4H), 3.97(s., 4H), 3.76-3.83 (t, J=4.0 Hz, 4H), 3.54 (m, 2H), 3.48 (s, 6H), 2.48(t, J=5.6 Hz, 2H), 1.95-2.09 (m, 4H), 1.77 (m, 2H). LC-MS: m/z(M+H)=669.2

Compound 228

Step A: methyl 2-(5-(difluoromethoxy)pyridin-2-yl)acetate

To a stirred solution of compound 7 (500 mg, 3 mmol) in MeCN was addedCs₂CO₃ (1.4 g, 4.5 mmol) followed by sodium 2-chloro-2,2-difluoroacetate(685 mg, 4.5 mmol) at room temperature. The whole mixture was heated to80° overnight. LC-MS showed desired product. It was filtered, evaporatedand purified by a standard method.

¹H NMR (CHLOROFORM-d) δ: 8.44 (s, 1H), 7.48 (dd, J=8.6, 2.7 Hz, 1H),7.34 (d, J=8.6 Hz, 1H), 6.55 (t, 1H, J=76 Hz), 3.88 (s, 2H), 3.74 (s,3H). LC-MS: m/z (M+H)=218.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-(difluoromethoxy)pyridin-2-yl)acetamide)(Compound 228)

The procedure was the same as Step B of Compound 84

¹H NMR (METHANOL-d₄) δ: 8.46 (d, J=2.4 Hz, 2H), 7.65 (dd, J=8.6, 2.4

Hz, 2H), 7.53 (d, J=8.9 Hz, 2H), 6.83 (t, J=72 Hz, 2H), 3.86 (s, 4H),3.50 (m, 2H), 2.40 (t, J=5.6 Hz, 2H), 1.97 (m, 4H), 1.71 (tm, 2H).LC-MS: m/z (M+H)=653.1

Compound 229

Step A: methyl 2-(6-cyanopyridin-2-yl)acetate

To a solution of 6-methylpicolinonitrile (2 g, 16.9 mmol) in THF (20 ml)was added slowly LiHMDS (17 ml, 1M in THF) at −78° C. under N2. Then thereaction mixture was stirred for 1 h at −78° C. and then dimethylcarbonate (1.52 g, 16.9 mmol) was added at −78° C. The mixture wasstirred for 30 mins at −78° C. and allowed to 0° C. for 30 mins.Saturated ammonium chloride was added to neutralize to adjust to PH=7-8,the mixture was extracted by ethyl acetate (100 ml*3), the organic layerwas dried by sodium sulfate, filtered, concentrated under vacuo to givethe residue. The residue was purified by a standard method to give thedesired product.

¹H NMR (CHLOROFORM-d): 7.84 (t, J=7.8 Hz, 1H), 7.64 (d, J=7.3 Hz, 1H),7.58 (d, J=8.1 Hz, 1H), 3.93 (s, 2H), 3.76 (s, 3H). LC-MS: m/z(M+H)=177.3

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-cyanopyridin-2-yl)acetamide)(Compound 229)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 7.84 (t, J=7.8 Hz, 2H), 7.71 (d, J=8.1 Hz, 2H),7.62 (d, J=7.8 Hz, 2H), 4.35 (s, 4H), 3.55-3.63 (m, 2H), 2.45 (t, J=5.8Hz, 2H), 2.06 (m, 2H), 1.93-2.01 (m, 2H), 1.74 (m, 2H). LC-MS: m/z(M+H)=571.5

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyrimidin-4-yl)acetamide)(Compound 230)

The procedure was the same as Compound 84

¹H NMR (CHLOROFORM-d) δ: 9.11-9.24 (s, 2H), 8.72 (d, J=5.1 Hz, 2H),7.42-7.59 (d, J=5.1 Hz, 2H), 4.24 (s, 4H), 3.59 (m, 2H), 2.45 (t, J=5.6Hz 0.2H), 1.89-2.13 (m, 4H), 1.74 (m, 2H). LC-MS: m/z (M+H)=523.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridazin-3-yl)acetamide)(Compound 231)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 9.16 (dd, J=4.8, 1.9 Hz, 2H), 7.72-7.75 (dd, J=4.8,1.9 Hz, 2H), 7.67-7.71 (dd, J=4.8, 1.9 Hz, 2H), 4.25 (s, 4H), 3.43-3.61(m, 2H), 2.31 (t, J=5.5 Hz, 2H), 1.93 (m, 2H), 1.84 (m, 2H), 1.49-1.70(m, 2H). LC-MS: m/z (M+H)=523.7

N,N′-(5,5′-(trans)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyrazin-2-yl)acetamide)(Compound 76)

The procedure was the same as Step B of Compound 84

¹H NMR (CDCl₃, 400 MHz) δ 8.73 (s, 2H), 8.57 (d, J=17.2 Hz, 4H), 4.26(s, 4H), 3.60-3.57 (m, 2H), 2.46 (t, J=5.6 Hz, 2H), 2.04 (m, 2H), 1.96(m, 2H), 1.75-1.73 (m, 2H). LC-MS: m/z (M+H)=523.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(thiazol-2-yl)acetamide)(Compound 100)

¹H NMR (CDCl₃, 400 MHz) δ: 7.76 (d, J=2.3 Hz, 2H), 7.37 (d, J=2.3 Hz,2H), 3.53 (s, 4H), 3.40 (m, 2H), 2.42 (t, J=5.0 Hz, 2H), 1.99 (m, 2H),1.93 (m, 2H), 1.72 (m, 2H). LC-MS: m/z (M+H)=533.6

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-aminopyridin-2-yl)acetamide)(Compound 232)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.70 (s, 2H), 7.25-7.38 (dd, J=7.3, 8.3 Hz 2H),6.47 (d, J=7.3 Hz, 2H), 6.33 (d, J=8.3 Hz, 2H), 5.88 (s, 4H), 3.72 (s,4H), 3.45-3.52 (m, 2H), 2.25-2.34 (t, J=5.4 Hz, 2H), 1.89-2.00 (m, 2H),1.83 (m, 2H), 1.62 (m, 2H). LC-MS: m/z (M+H)=551.7

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-aminopyridin-2-yl)acetamide)(Compound 233)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.61 (s, 2H), 7.84 (d, J=2.4 Hz, 2H), 7.02 (d,J=8.1 Hz, 2H), 6.89 (dd, J=8.3, 2.7 Hz, 2H), 5.23 (s, 4H), 3.78 (s, 4H),3.48 (m, 2H), 2.29 (t, J=5.4 Hz, 2H), 1.93 (m, 2H), 1.85 (m, 2H), 1.62(m, 2H). LC-MS: m/z (M+H)=551.8

N,N′-(5,5′-(trans-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-cyanopyridin-2-yl)acetamide)(Compound 113)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d6) δ: 12.90 (br. s., 2H), 8.78 (dd, J=5.0, 1.7 Hz, 2H),8.35 (dd, J=7.9, 1.7 Hz, 2H), 7.56 (dd, J=7.9, 5.0 Hz, 2H), 4.19 (s,4H), 3.50 (dt, J=11.1, 5.6 Hz, 2H), 2.31 (t, J=5.5 Hz, 2H), 1.89-2.03(m, 2H), 1.77-1.89 (m, 2H), 1.54-1.66 (m, 2H). LC-MS: m/z (M+H)=571.7

Compound 234

Step A: 6-(2-methoxy-2-oxoethyl)nicotinic acid

The procedure is the same as step A of Compound 84

LC-MS: m/z (M−H)=194.1

Step B: methyl 2-(5-(dimethylcarbamoyl)pyridin-2-yl)acetate

A solution of 6-(2-methoxy-2-oxoethyl)nicotinic acid (37 mg, 0.19 mmol),HATU (72.6 mg, 0.19 mmol), and N-ethyl-N-isopropylpropan-2-amine (26.5mg, 0.20 mmol) in N,N-dimethylformamide (2 ml) was stirred at roomtemperature for 15 min, then, dimethyl amine (0.05 mL, 1 mol solution inTHF) was added and continued to stir overnight. The reaction is quenchedwith water, extracted with EtOAc and purified by a standard method togive the desired compound.

¹H NMR (CHLOROFORM-d) δ: 8.65 (d, J=1.9 Hz, 1H), 7.79 (dd, J=7.9, 2.3Hz, 1H), 7.40 (d, J=8.1 Hz, 1H), 3.92 (s, 2H), 3.75 (s, 3H), 3.15 (s,3H), 3.04 (s, 3H). LC-MS: m/z (M+H)=223.1.

Step C:6-[[5-[3-[5-[[2-[5-(dimethylcarbamoyl)pyridin-2-yl]acetyl]amino]-1,3,4-thiadiazol-2-yl]cyclohexyl]-1,3,4-thiadiazol-2-yl]carbamoylmethyl]-N,N-dimethyl-pyridine-3-carboxamide(Compound 234)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 8.75 (s, 2H), 7.88 (d, J=6.2 Hz, 2H), 7.48 (d,J=7.8 Hz, 2H), 4.20 (s, 4H), 3.55-3.62 (m, 2H), 3.16 (s, 6H), 3.06 (s,6H), 2.47 (t, J=5.4 Hz, 2H), 2.01-2.07 (m, 2H), 1.92-1.99 (m, 2H),1.72-1.79 (m, 2H). LC-MS: m/z (M+H)=663.3.

Compound 105

Step A: ethyl 2-(6-(dimethylamino)pyridin-2-yl)acetate

To the solution of ethyl 2-(6-aminopyridin-2-yl)acetate (150 mg, 0.83mmol) and paraformaldehyde (54.8 mg, 1.83 mmol) in methanol (6 mL) wasadded NaBH₃CN (130.3 mg, 2.08 mmol) and AcOH (1 drop, cat.). The mixturewas stirred at room temperature for 12 h. Then, the reaction wasquenched with aqueous ammonium chloride and extracted with DCM. Theorganic layer was with brine, dried over sodium sulfate and evaporatedunder reduced pressure. The residue was purified by a standard method toget desired product (100 mg).

¹H NMR (CHLOROFORM-d) δ: 7.41 (t, J=7.8 Hz, 1H), 6.52 (d, J=7.3 Hz, 1H),6.40 (d, J=8.6 Hz, 1H), 4.20 (q, J=7.3 Hz, 2H), 3.70 (s, 2H), 3.08 (s,6H), 1.24-1.32 (t, J=7.3 Hz, 3H). LC-MS: m/z (M+H)=208.6

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(dimethylamino)pyridin-2-yl)acetamide)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.67 (s, 2H), 7.46 (t, J=7.9 Hz, 2H), 6.52 (d,J=8.6 Hz, 2H), 6.55 (d, J=7.3 Hz, 2H), 3.81 (s, 4H), 3.49 (m, 2H), 2.98(s, 12H), 2.32 (d, J=5.4 Hz, 2H), 1.94 (m, 2H), 1.78-1.89 (m, 2H), 1.63(m, 2H). LC-MS: m/z (M+H)=607.8

Compound 115

Step A: methyl 2-(3-aminopyridin-2-yl)acetate

Methyl 2-(3-nitropyridin-2-yl)acetate (1 g, 5 mmol) was dissolved inMeOH and stirred under the atmosphere of H2 at room temperatureovernight. LC-MS find the desired product. It was filtered through a padof Celite and evaporated to get the desired product. It was directlyused for the next step. LC-MS: m/z (M+H)=167.1

Step B: methyl 2-(3-(dimethylamino)pyridin-2-yl)acetate

To the solution of methyl 2-(5-aminopyridin-2-yl)acetate (600 mg, 3.6mmol) and paraformaldehyde (578.3 mg, 19.3 mmol) in methanol (20 mL) wasadded NaBH3CN (1.2 g, 19.3 mmol) and AcOH (1 drop, cat.). The mixturewas stirred at room temperature for 12 h. Then, the reaction wasquenched with aqueous ammonium chloride and extracted with DCM. Theorganic layer was with brine, dried over sodium sulfate and evaporatedunder reduced pressure. The residue was purified by a standard method toget desired product. LC-MS: m/z (M+H)=195.1

Step C:N,N′-(5,5′-(trans-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(dimethylamino)pyridin-2-yl)acetamide)(Compound 115)

The procedure was the same as Step B of Compound 84

¹H NMR (METHANOL-d4) δ: 8.24 (d, J=3.8 Hz, 2H), 7.72 (d, J=8.3 Hz, 2H),7.37 (dd, J=8.1, 4.8 Hz, 2H), 4.61 (s, 4H), 3.57 (m, 2H), 2.71 (s, 12H),2.45-2.52 (t, J=5.4 Hz, 2H), 1.93-2.15 (m, 4H), 1.69-1.83 (m, 2H).LC-MS: m/z (M+H)=607.8

Compound 235 and Compound 236

The procedure was the same as Compound 105

Step A

To the solution of methyl 2-(3-aminophenyl)acetate (150 mg, 0.83 mmol)and acetaldehyde (80.5 mg, 1.83 mmol) in methanol (6 mL) was addedNaBH3CN (130.3 mg, 2.08 mmol) and AcOH (1 drop, cat.). The mixture wasstirred at room temperature for 12 h. Then, the reaction was quenchedwith aqueous ammonium chloride and extracted with DCM. The organic layerwas with brine, dried over sodium sulfate and evaporated under reducedpressure. The residue was purified by a standard method to get methyl2-(3-(diethylamino)phenyl)acetate and methyl2-(3-(ethylamino)phenyl)acetate.

methyl 2-(3-(diethylamino)phenyl)acetate

LC-MS: m/z (M+H)=222.4

methyl 2-(3-(ethylamino)phenyl)acetate

LC-MS: m/z (M+H)=194.4

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(diethylamino)phenyl)acetamide)(Compound 235)

The procedure was the same as Step B of Compound 105.

¹H NMR (DMSO-d₆) δ: 12.60 (s, 2H), 7.07 (t, J=7.9 Hz, 2H), 6.65 (s, 2H),6.46-6.57 (m, 4H), 3.65 (s, 4H), 3.47 (m, 2H), 3.31 (q, J=7.0 Hz, 8H),2.28 (t, J=5.5 Hz, 2H), 1.87-1.99 (m, 2H), 1.71-1.87 (m, 2H), 1.52-1.66(m, 2H), 1.07 (t, J=7.0 Hz, 12H). LC-MS: m/z (M+H)=661.9

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(ethylamino)phenyl)acetamide)(Compound 236)

¹H NMR (DMSO-d₆) δ: 12.65 (s, 2H), 7.01 (t, J=7.8 Hz, 2H), 6.51 (s, 2H),6.43 (d, J=8.1 Hz, 2H), 6.47 (dd, J=7.5, 8.1 Hz, 2H), 5.55 (t, J=5.5 Hz,2H), 3.64 (s, 4H), 3.41-3.52 (m, 2H), 2.94-3.07 (m, 4H), 2.29 (t, J=5.6Hz, 2H), 1.92 (d, J=4.8 Hz, 2H), 1.80-1.89 (m, 2H), 1.61 (d, J=5.6 Hz,2H), 1.14 (t, J=7.1 Hz, 6H). LC-MS: m/z (M+H)=605.9

Compound 237

The procedure was the same as Compound 105

Step A: ethyl 2-(4-(diethylamino)phenyl)acetate

The procedure was the same as Step B of Compound 105.

¹H NMR (CHLOROFORM-d) δ: 7.15 (d, J=8.3 Hz, 2H), 6.66 (d, J=7.3 Hz, 2H),4.16 (q, J=7.0 Hz, 2H), 3.52 (s, 2H), 3.36 (q, J=7.1 Hz, 4H), 1.24-1.31(t, J=7.0 Hz, 3H), 1.17 (t, J=7.1 Hz, 6H). LC-MS: m/z (M+H)=236.5

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(diethylamino)phenyl)acetamide)(Compound 237)

¹H NMR (DMSO-d₆) δ: 12.60 (s, 2H), 7.10 (d, J=8.6 Hz, 4H), 6.54-6.65 (d,J=8.5 Hz, 4H), 3.60 (s, 4H), 3.46 (m, 2H), 3.29 (q, J=6.9 Hz, 8H), 2.28(t, J=5.5 Hz, 2H), 1.87-1.98 (m, 2H), 1.77-1.87 (m, 2H), 1.55-1.64 (m,2H), 1.02-1.08 (t, J=7.0 Hz, 12H). LC-MS: m/z (M+H)=661.9

Compound 104

Step A: ethyl 2-(6-acetamidopyridin-2-yl)acetate

To the solution of ethyl 2-(6-aminopyridin-2-yl)acetate (50 mg, 0.28mmol) and N-ethyl-N-isopropylpropan-2-amine (71.0 mg, 0.55 mmol) in DCMwas added acetyl chloride (43.2 mg, 0.55 mmol) dropwise at roomtemperature. The mixture was continued to stir for 1 h, washed withbrine and evaporated in vacuum to dryness. The residue was purified by astandard method to afford desired compound. LC-MS: m/z (M+H)=222.4

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-acetamidopyridin-2-yl)acetamide)(Compound 104)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.71 (s, 2H), 10.42 (s, 2H), 7.97 (d, J=8.6 Hz,2H), 7.74 (t, J=7.8 Hz, 2H), 7.09 (d, J=7.3 Hz, 2H), 3.94 (s, 4H), 3.49(m, 2H), 2.31 (d, J=5.4 Hz, 2H), 2.07 (s, 6H), 1.94 (m, 2H), 1.85 (m,2H), 1.62 (m, 2H). LC-MS: m/z (M+H)=635.8

Compound 238

Step A: ethyl 2-(4-(methylsulfonamido)phenyl)acetate

The solution of ethyl 2-(4-aminophenyl)acetate (500 mg, 2.79 mmol) andtriethylamine (0.58 mL, 4.18 mmol) in dichloromethane (30 mL) was addedmethanesulfonyl chloride (0.24 mL, 3.07 mmol) dropwise at roomtemperature and stirred overnight. The mixture was washed with brine,dried over sodium sulfate and evaporated under reduced pressure. Theresidue was purified by a standard method to give desired compound.LC-MS: m/z (M+H)=258.3

Step B: 2-(4-(methylsulfonamido)phenyl)acetic acid

The solution of ethyl 2-(4-(methylsulfonamido)phenyl)acetate (200 mg,0.77 mmol) and lithium hydroxide hydrate (130.4 mg, 3.11 mmol) inMeOH/H₂O (10 mL, 1:1) was stirred at room temperature for 12 h. Thereaction mixture was evaporated under reduced pressure. The residue wasused for the next step without purification. LC-MS: m/z (M+H)=230.4

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(methylsulfonamido)phenyl)acetamide)(Compound 238)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.70 (br. s., 2H), 9.71 (br. s., 2H), 7.29 (d,J=8.6 Hz, 4H), 7.17 (d, J=8.6 Hz, 4H), 3.76 (s, 4H), 3.43-3.52 (m, 2H),2.94-3.00 (m, 6H), 2.29 (t, J=5.6 Hz, 2H), 1.78-1.98 (m, 4H), 1.56-1.67(m, 2H). LC-MS: m/z (M+H)=705.7

Compound 239

Step A: ethyl 2-(4-((tert-butoxycarbonyl)amino)phenyl)acetate

A solution of di-tert-butyl dicarbonate (387 mg, 1.77 mmol) in toluene(5 ml) was added a vessel containing ethyl 2-(4-aminophenyl)acetate (288mg, 1.61 mmol), the reaction mixture was heated at 85° C. for 4 h. LCMSshowed that the desired product was detected, the mixture wasconcentrated to give the residue, the residue was purified by a standardmethod to give the desired product. LC-MS: m/z (M+H)=280.3

Step B:di-tert-butyl((((5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl))bis(2-oxoethane-2,1-diyl))bis(4,1-phenylene))dicarbamate

A solution of5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (68 mg,0.241 mmol), ethyl 2-(4-(tert-butoxycarbonylamino)phenyl)acetate (201mg, 0.72 mmol), and cesium carbonate (251 mg, 0.771 mmol) inN,N-dimethylformamide (2 ml) was heated to 130° C. for 45 mins undermicrowave. Then the reaction mixture was cooled to room temperature andwas poured into to water. The mixture was extracted by ethyl acetate (50ml*3), the organic layer was washed by brine, dried by sodium sulfate,filtered, concentrated to give the residue; the residue was purified bya standard method to give the desired product.

¹H NMR (CHLOROFORM-d) δ: 7.36 (br, 8H), 3.96 (s, 4H), 3.53-3.57 (m, 2H),2.40 (t, J=5.6 Hz, 2H), 1.97-2.06 (m, 4H), 1.77 (m, 2H), 1.52 (s, 18H).LC-MS: m/z (M+H)=750.0.

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-aminophenyl)acetamide)(Compound 239)

To a solution of tert-butyl4,4′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(4,1-phenylene)dicarbamate(20 mg) in DCM (2 ml) was added TFA (0.5 mL). The reaction mixture wasconcentrated after 0.5 h to give the desired product.

¹H NMR (METHANOL-d₄) δ: 7.09 (d, J=8.3 Hz, 4H), 6.70-6.74 (d, J=8.3 Hz,4H), 3.68 (s, 4H), 3.53-3.57 (m, 2H), 2.43 (t, J=5.6 Hz, 2H), 1.97-2.06(m, 4H), 1.72-1.77 (m, 2H). LC-MS: m/z (M+H)=549.7.

Compound 240

The procedure was the same as Compound 241

Step A: dimethyl 2-(5-fluoropyridin-2-yl)malonate

The solution of 2-bromo-5-fluoropyridine (1.0 g, 5.68 mmol), dimethylmalonate (3.0 g, 22.7 mmol) picolinic acid (559.6 mg, 4.54 mmol), CuI(431.8 mg, 2.27 mmol) and Cs₂CO₃ (5.6 g, 17.05 mmol) in DMF (30 mL) wasstirred at 100 degree for 12 h. After cooling to room temperature, thereaction mixture was filtered, diluted with H₂O and extracted with ethylacetate. The organic layer was washed with brine, dried over sodiumsulfate and evaporated under reduced pressure. The residue was purifiedby a standard method to give desired compound (400 mg). LC-MS: m/z(M+H)=228.2

Step B: methyl 2-(5-fluoropyridin-2-yl)acetate

The solution of dimethyl 2-(5-fluoropyridin-2-yl)malonate (400 mg 1.76mmol), NaCl (109.2 mg, 1.87 mmol) and H₂O (56.3 g, 3.13 mmol) in DMSO (3mL) was stirred at 130 degree for 6 h. After cooling to roomtemperature, the reaction mixture was diluted with H₂O and extractedwith ethyl acetate. The organic layer was dried over sodium sulfate andevaporated under reduced pressure. The residue was purified by astandard method to get desired product. LC-MS: m/z (M+H)=170.1

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-fluoropyridin-2-yl)acetamide)(Compound 240)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.74 (br. s., 2H), 8.50 (d, J=3.0 Hz, 2H), 7.72(td, J=8.7, 3.0 Hz, 2H), 7.49 (dd, J=8.7, 4.4 Hz, 2H), 4.03 (s, 4H),3.43-3.55 (m, 2H), 2.31 (t, J=5.4 Hz, 2H), 1.90-2.00 (m, 2H), 1.79-1.89(m, 2H), 1.56-1.67 (m, 2H). LC-MS: m/z (M+H)=557.6

Compound 101 and Compound 242

The procedure was the same as Compound 240 for steps A-C

Step A: dimethyl 2-(5-cyanopyridin-2-yl)malonate

LC-MS: m/z (M+H)=235.4

Step B: methyl 2-(5-cyanopyridin-2-yl)acetate

LC-MS: m/z (M+H)=177.3

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-cyanopyridin-2-yl)acetamide)(Compound 101)

¹H NMR (DMSO-d₆) δ: 12.80 (br. s., 2H), 8.97 (s, 2H), 8.24-8.37 (d,J=8.1 Hz, 2H), 7.59-7.73 (d, J=8.3 Hz, 2H), 4.15 (s, 4H), 3.49 (m, 2H),2.32 (t, J=5.8 Hz, 2H), 1.94 (m, 2H), 1.85 (m., 2H), 1.62 (m, 2H).LC-MS: m/z (M+H)=571.7

Step D:6,6′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl))bis(2-oxoethane-2,1-diyl))dinicotinamide(Compound 242)

To a solution ofN,N-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-cyanopyridin-2-yl)acetamide)(30 mg, 0.053 mmol) in DMSO (1 ml) was added sodium hydroxide solution(4 M, 0.1 mL) at 0° C. Then the reaction mixture was stirred for 2 minsand then hydrogen peroxide (30% in water, 1 ml) was added. The mixturewas stirred for 10 mins TLC indicated that the starting material wasconsumed. The mixture was purified by a standard method to give thedesired product.

¹H NMR (METHANOL-d₄) δ: 9.00 (d, J=1.9 Hz, 2H), 8.27 (dd, J=8.2, 2.3 Hz,2H), 7.58 (d, J=8.3 Hz, 2H), 4.06-4.20 (s, 4H), 3.50-3.63 (m, 2H), 2.45(t, J=5.8 Hz, 2H), 1.95-2.11 (m, 4H), 1.70-1.82 (m, 2H); LC-MS: m/z(M+H)=607.5

Compound 243

Step A: (6-(2-methoxy-2-oxoethyl)pyridin-3-yl)methanaminium chloride

To a solution of methyl 2-(5-cyanopyridin-2-yl)acetate (1.2 g, 6.8 mmol)in methanol (20 ml) was added hydrogen chloride in methanol (4 M, 20ml), and then Pd/C (200 mg). Then the reaction mixture was hydrogenatedunder hydrogen atmosphere overnight. The reaction mixture was filtered,the filtrate was concentrated to give the crude product and was washedby ethyl acetate to give the desired product. LC-MS: m/z (M+H)=181.5

Step B: methyl2-(5-((tert-butoxycarbonylamino)methyl)pyridin-2-yl)acetate

To a solution of (6-(2-methoxy-2-oxoethyl)pyridin-3-yl)methanaminiumchloride (400 mg, 2.22 mmol) in CH₂Cl₂ (50 ml) was added triethylamine(561 mg, 5.55 mmol), and then was added (Boc)₂O (726 g, 3.33 mmol). Thereaction mixture was stirred overnight. LCMS showed that the desiredproduct was detected, the mixture was concentrated to give the residue,the residue was purified by a standard method to give the desiredproduct.

¹H NMR (CHLOROFORM-d) δ: 8.49 (d, J=1.9 Hz, 1H), 7.63 (d, J=6.4 Hz, 1H),7.28 (t, J=4.0 Hz, 1H), 4.33 (d, J=5.6 Hz, 2H), 3.86 (s, 2H), 3.74 (s,3H). LC-MS: m/z (M+H)=281.5

Step C: tert-butyl(6,6′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(pyridine-6,3-diyl))bis(methylene)dicarbamate

The procedure is the same as Step B of Compound 84 LC-MS: m/z(M+H)=779.5

Step D:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-(aminomethyl)pyridin-2-yl)acetamide)(Compound 243)

To a solution of tert-butyl(6,6′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(pyridine-6,3-diyl))bis(methylene)dicarbamate(20 mg, 0.026 mmol) was added hydrogen chloride solution in methanol(4M, 10 ml) and the resulting solution was stirred for 1 h. LCMS showedthe starting material was consumed and the desired product was detected.The mixture was concentrated to give the desired product.

¹H NMR (METHANOL-d₄) δ: 9.10 (s, 2H), 8.68-8.82 (d, J=8.3 Hz, 2H),8.12-8.28 (d, J=8.3 Hz, 2H), 4.48 (s, 4H), 3.60-3.64 (m, 6H), 2.45 (t,J=5.4 Hz, 2H), 2.05-2.12 (m, 2H), 1.99 (m, 2H), 1.75 (m, 2H). LC-MS: m/z(M+H)=579.5

Compound 244

The procedure was the same as Compound 243

Step A to Step B: methyl2-(6-((tert-butoxycarbonylamino)methyl)pyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.65 (t, J=7.7 Hz, 1H), 7.14-7.23 (m, 2H), 5.59(br. s., 1H), 4.44 (d, J=5.1 Hz, 2H), 3.86 (s, 2H), 3.74 (s, 3H), 1.48(s, 9H); LC-MS: m/z (M+H)=281.5.

Step C to Step D:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(aminomethyl)pyridin-2-yl)acetamide)(Compound 244)

¹H NMR (METHANOL-d₄) δ: 7.92 (t, J=7.8 Hz, 2H), 7.50 (d, J=7.8 Hz, 2H),7.44 (d, J=7.8 Hz, 2H), 4.28-4.36 (m, 4H), 3.56-3.67 (m, 6H), 2.46 (t,J=5.8 Hz, 2H), 2.07 (d, J=5.9 Hz, 2H), 1.94-2.01 (m, 2H), 1.73-1.79 (m,2H); LC-MS: m/z (M+H)=579.5

Compound 245

Step A: methyl 2-(3-cyanophenyl)acetate

To a solution of 2-(3-cyanophenyl)acetic acid (1, 6.2 mmol) in methanol(20 ml) was added sulfurous dichloride (5 ml) and 0.1 mlN,N-dimethylformamide. Then the reaction mixture was heated to 80° C.for 2 h. When TLC indicated that the starting material was consumed, themixture was concentrated to give the residue. The residue was pouredinto water (20 ml) and was extracted by ethyl acetate (50 m1*2). Theorganic layer was dried by anhydrous sodium sulfate, filtered,concentrated to give the desired product. LC-MS: m/z (M+H)=176.5

Step B: (3-(2-methoxy-2-oxoethyl)phenyl)methanaminium chloride

The procedure is the same as Step A of Compound 243

¹H NMR (METHANOL-d₄) δ: 7.33-7.47 (m, 4H), 3.73 (s, 2H), 3.70 (s, 3H),3.37 (s, 2H). LC-MS: m/z (M+H)=180.5

Step C: methyl 2-(3-((tert-butoxycarbonylamino)methyl)phenyl)acetate

The procedure is the same as Step B of Compound 243

¹H NMR (CHLOROFORM-d) δ: 7.14-7.26 (m, 4H), 4.87 (br. s., 1H), 4.33 (d,J=5.4 Hz, 2H), 3.72 (s, 3H), 3.64 (s, 2H), 1.48 (s, 9H). LC-MS: m/z(M+H)=280.5

Step D to Step E: tert-butyl(3,3′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(3,1-phenylene))bis(methylene)dicarbamate

The procedure is the same as Step B to Step C of Compound 238

LC-MS: m/z (M+H)=777.5

Step F:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(aminomethyl)phenyl)acetamide)(Compound 245)

The procedure is the same as Step D of Compound 243

¹H NMR (METHANOL-d₄) δ: 7.34-7.52 (m, 8H), 4.09-4.18 (m, 4H), 3.93 (s,4H), 3.62 (m, 2H), 2.45 (t, J=5.5 Hz, 2H), 2.04-2.13 (m, 4H), 1.70-1.81(m, 2H). LC-MS: m/z (M+H)=577.5

Compound 241

Step A: dimethyl 2-(5-nitropyridin-2-yl)malonate

The solution of dimethyl 2-(5-nitropyridin-2-yl)malonate (6.0 g, 37.8mmol), dimethyl malonate (10.0 g, 75.7 mmol) and Cs₂CO₃ (24.7 g, 75.7mmol) in DMF (50 mL) was stirred at 100 degree for 12 h. After coolingto room temperature, the reaction mixture was filtered, diluted with H₂Oand extracted with ethyl acetate. The organic layer was washed withbrine, dried over sodium sulfate and evaporated under reduced pressureto get crude product. LC-MS: m/z (M+H)=255.3

Step B: methyl 2-(5-nitropyridin-2-yl)acetate

The solution of dimethyl 2-(5-nitropyridin-2-yl)malonate (10.0 g 41.7mmol), NaCl (2.58 g, 44.20 mmol) and H₂O (1.3 g, 74.23 mmol) in DMSO (50mL) was stirred at 130 degree for 6 h. After cooling to roomtemperature, the reaction mixture was diluted with H₂O and extractedwith ethyl acetate. The organic layer was dried over sodium sulfate andevaporated under reduced pressure. The residue was purified by astandard method to get desired product.

¹H NMR (CHLOROFORM-d) δ: 9.40 (d, J=2.4 Hz, 1H), 8.48 (dd, J=8.6, 2.7Hz, 1H), 7.56 (d, J=8.6 Hz, 1H), 4.02 (s, 2H), 3.77 (s, 3H). LC-MS: m/z(M+H)=197.1

Step C: methyl 2-(5-aminopyridin-2-yl)acetate

The solution of methyl 2-(5-nitropyridin-2-yl)acetate (500 mg, 2.5 mmol)and Pd/C (50 mg) in methanol (20 mL) was stirred under H₂ at roomtemperature for 3 h. Then, the reaction mixture was filtered andevaporated under reduced pressure to get desired product for the nextstep without further purification.

¹H NMR (CHLOROFORM-d) δ: 8.05 (d, J=2.7 Hz, 1H), 7.08 (d, J=8.3 Hz, 1H),6.98 (dd, J=8.2, 2.8 Hz, 1H), 3.75 (s, 2H), 3.72 (s, 3H). LC-MS: m/z(M+H)=167.3

Step D: methyl 2-(5-(dimethylamino)pyridin-2-yl)acetate

To the solution of methyl 2-(5-aminopyridin-2-yl)acetate (800 mg, 4.8mmol) and paraformaldehyde (578.3 mg, 19.3 mmol) in methanol (20 mL) wasadded NaBH₃CN (1.2 g, 19.26 mmol) and AcOH (1 drop, cat.). The mixturewas stirred at room temperature for 12 h. Then, the reaction wasquenched with aqueous ammonium chloride and extracted with DCM. Theorganic layer was with brine, dried over sodium sulfate and evaporatedunder reduced pressure. The residue was purified by a standard method toget desired product.

¹H H NMR (CHLOROFORM-d) δ: 8.09 (d, J=3.2 Hz, 1H), 7.14 (d, J=8.6 Hz,1H), 6.99 (dd, J=8.6, 3.2 Hz, 1H), 3.77 (s, 2H), 3.72 (s, 3H), 2.98 (s,6H). LC-MS: m/z (M+H)=195.2

Step E:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-(dimethylamino)pyridin-2-yl)acetamide)(Compound 241)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 8.02 (d, J=3.0 Hz, 2H), 7.16 (d, J=8.6 Hz, 2H),6.98 (dd, J=8.6, 3.2 Hz, 2H), 3.49 (dt, J=11.4, 5.7 Hz, 2H), 3.04-3.34(m, 4H), 2.95 (s, 12H), 2.39 (t, J=5.6 Hz, 2H), 1.87-2.05 (m, 4H),1.65-1.77 (m, 2H). LC-MS: m/z (M+H)=607.7

Compound 246

The procedure is the same as Step B to Step F of Compound 245

Step A: (4-(2-methoxy-2-oxoethyl)phenyl)methanaminium chloride

¹H NMR (DMSO-d₆) δ: 8.47 (br. s., 2H), 7.45 (d, J=7.8 Hz, 2H), 7.30 (d,J=7.5 Hz, 2H), 3.98 (br. s., 2H), 3.71 (s, 2H), 3.71 (s, 3H).

Step B to Step C: 2-(4-((tert-butoxycarbonylamino)methyl)phenyl)aceticacid

¹H NMR (DMSO-d₆) δ: 7.34 (t, J=6.0 Hz, 1H), 7.11-7.20 (m, J=7.8 Hz, 2H),7.00-7.10 (m, J=7.8 Hz, 2H), 4.05 (d, J=6.2 Hz, 2H), 3.20 (s, 2H), 1.38(s, 9H)

Step D to Step F:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(aminomethyl)phenyl)acetamide)(Compound 246)

¹H NMR (METHANOL-d₄) δ: 7.42-7.50 (m, 8H), 4.09-4.15 (m, 4H), 3.90 (s,4H), 3.57-3.65 (m, 2H), 2.45 (t, J=5.6 Hz, 2H), 2.05-2.13 (m, 2H),1.92-2.02 (m, 2H), 1.70-1.80 (m, 2H); LC-MS: m/z (M+H)=577.5

Compound 247

Step A: ethyl 2-(4-ethoxypyridin-2-yl)acetate

The solution of 2-(4-ethoxypyridin-2-yl)acetonitrile (150 mg, 0.92 mmol)in EtOH/HCl (6 mL/2 mL) was stirred at 70 degree for 2 h. The mixturewas evaporated in vacuum. The residue was used for the next step withoutfurther purification. LC-MS: m/z (M+H)=210.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-ethoxypyridin-2-yl)acetamide)(Compound 247)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 8.31 (d, J=5.6 Hz, 2H), 6.92 (d, J=2.1 Hz, 2H),6.80 (dd, J=5.8, 2.0 Hz, 2H), 4.32 (br. s., 4H), 4.11 (q, J=6.9 Hz, 4H),3.46-3.56 (m, 2H), 2.35-2.45 (m, 2H), 1.85-2.03 (m, 4H), 1.67-1.76 (m,2H), 1.42 (t, J=7.0 Hz, 6H). LC-MS: m/z (M+H)=609.8

Compound 248

Step A: 2-(2-fluoro-5-methoxyphenyl)acetic acid

The solution of 2-(2-fluoro-5-methoxyphenyl)acetonitrile (200 mg, 1.0mmol) and sodium hydroxide (81.5 mg, 2.0 mmol) in water was stirred at100 degree for 8 h. The reaction mixture was evaporated under reducedpressure to get the crude product for the next step without furtherpurification. LC-MS: m/z (M−H)=183.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-fluoro-5-methoxyphenyl)acetamide)(Compound 248)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.74 (s, 2H), 7.07-7.16 (m, 2H), 6.97 (dd, J=5.9,3.2 Hz, 2H), 6.87 (dt, J=8.9, 3.6 Hz, 2H), 3.86 (s, 4H), 3.68-3.77 (s,8H), 3.44-3.53 (m, 2H), 2.30 (t, J=5.4 Hz, 2H), 1.89-2.02 (m, 2H),1.76-1.89 (m, 2H), 1.62 (m, 2H). LC-MS: m/z (M+H)=615.7

Compound 249

Step A: methyl 2-(6-(3-hydroxyazetidin-1-yl)pyridin-2-yl)acetate

The solution of methyl 2-(6-bromopyridin-2-yl)acetate (500.0 mg, 2.17mmol), azetidin-3-ol hydrochloride (285.9 mg, 2.61 mmol), CuI (248.3 mg,1.30 mmol), L-proline (149.7 mg, 1.30 mmol) and Cs₂CO₃ (24.7 g, 75.7mmol) in DMSO (6 mL) was stirred at 90 degree for 12 h under N₂. Aftercooling to room temperature, the reaction mixture was filtered, dilutedwith H₂O and extracted with ethyl acetate. The organic layer was washedwith brine, dried over sodium sulfate and evaporated under reducedpressure. The residue was purified by a standard method to give desiredcompound.

¹H NMR (CHLOROFORM-d) δ: 7.43 (dd, J=8.1, 7.5 Hz, 1H), 6.59 (d, J=7.3Hz, 1H), 6.21 (d, J=8.3 Hz, 1H), 4.75 (tt, J=6.4, 4.6 Hz, 1H), 4.29 (dd,J=9.4, 6.4 Hz, 2H), 3.86 (dd, J=9.5, 4.4 Hz, 2H), 3.69-3.77 (m, 5H).LC-MS: m/z (M+H)=223.4

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3-hydroxyazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 249)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 7.32 (t, J=7.8 Hz, 2H), 6.43 (d, J=7.3 Hz, 2H),6.14 (d, J=8.3 Hz, 2H), 4.56-4.70 (m, 2H), 4.23 (t, J=7.5 Hz, 4H),3.77-3.88 (m, 4H), 3.64-3.74 (m, 2H), 3.36-3.47 (m, 2H), 2.31 (d, J=5.1Hz, 2H), 1.75-1.98 (m, 4H), 1.55-1.68 (m, 2H). LC-MS: m/z (M+H)=663.9

Compound 250

The procedure was the same as Compound 249.

Step A: methyl 2-(6-(3,3-difluorocyclobutylamino)pyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.40-7.50 (m, 1H), 6.65 (d, J=7.3 Hz, 1H), 6.26(d, J=8.3 Hz, 1H), 4.94 (br. s., 1H), 4.02-4.17 (m, 1H), 3.71-3.77 (m,3H), 3.66-3.71 (m, 2H), 3.00-3.15 (m, 2H), 2.39-2.60 (m, 2H). LC-MS: m/z(M+H)=257.6

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3,3-difluorocyclobutylamino)pyridin-2-yl)acetamide)(Compound 250)

¹H NMR (CHLOROFORM-d) δ: 7.41-7.49 (m, 2H), 6.57 (d, J=7.3 Hz, 2H), 6.29(d, J=8.3 Hz, 2H), 5.86 (d, J=5.4 Hz, 2H), 4.07-4.23 (m, 2H), 3.84 (s,4H), 3.52-3.58 (m, 2H), 3.08-3.25 (m, 4H), 2.51-2.63 (m, 4H), 2.46 (t,J=5.6 Hz, 2H), 1.88-2.11 (m, 4H), 1.70-1.82 (m, 2H). LC-MS: m/z(M+H)=731.5

Compound 251

The procedure was the same as Compound 249

Step A: methyl 2-(6-(3-fluoroazetidin-1-yl)pyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.46 (t, J=7.8 Hz, 1H), 6.65 (d, J=7.3 Hz, 1H),6.24 (d, J=8.3 Hz, 1H), 5.46-5.55 (m, 0.5H), 5.33-5.40 (m, 0.5H),4.26-4.39 (m, 2H), 4.05-4.20 (m, 2H), 3.71-3.77 (m, 5H). LC-MS: m/z(M+H)=211.6

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3-fluoroazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 251)

¹H NMR (DMSO-d₆) δ: 12.70 (br. s., 2H), 7.55 (t, J=7.0 Hz, 2H), 6.70 (d,J=7.3 Hz, 2H), 6.38 (d, J=7.5 Hz, 2H), 5.52-5.63 (m, 1H), 5.35-5.47 (m,1H), 4.18-4.42 (m, 4H), 3.91-4.09 (m, 4H), 3.86 (br. s., 4H), 3.45-3.54(m, 2H), 2.31 (br. s., 2H), 1.90-2.05 (m, 2H), 1.78-1.90 (m, 2H), 1.62(d, J=5.6 Hz, 2H). LC-MS: m/z (M+H)=667.8

Compound 252 and Compound 253

Step A: methyl 2-(5-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetate

The procedure is the same as Step B of Compound 254

¹H NMR (CHLOROFORM-d) δ: 7.88 (d, J=2.7 Hz, 1H), 7.23 (d, J=8.3 Hz, 1H),6.87 (dd, J=8.5, 2.8 Hz, 1H), 4.31 (t, J=11.6 Hz, 4H), 3.84 (s, 2H),3.74 (s, 3H). LC-MS: m/z (M+H)=243.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 252) andN-(5-((1S,3S)-3-(5-acetamido-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(5-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide(Compound 253)

A mixture of methyl 2-(5-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetate(100 mg),5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (50 mg,0.18 mmol), cesium carbonate (346.1 mg, 1.06 mmol) in DMA (3 mL) washeated to 130° C. under nitrogen atmosphere and microwave for 45 min.The mixture was evaporated in vacuum to dryness. The residue waspurified by a standard method to give Compound 252 and Compound 253.

Compound 252

¹H NMR (CHLOROFORM-d) δ: 7.81 (d, J=2.7 Hz, 2H), 7.25 (d, J=8.3 Hz, 2H),6.87 (dd, J=8.5, 2.8 Hz, 2H), 4.27 (t, J=11.7 Hz, 9H), 3.44-3.54 (m,2H), 2.29-2.49 (t, J=5.6 Hz, 2H), 1.97 (m, 2H), 1.91 (m, 2H), 1.72 (m,2H). LC-MS: m/z (M+H)=703.2

Compound 253

¹H NMR (CHLOROFORM-d) δ: 8.01 (s, 1H), 7.25 (d, J=8.3 Hz, 1H), 6.89 (d,J=6.7 Hz, 1H), 4.33 (t, J=11.6 Hz, 4H), 4.04 (s, 2H), 3.59 (m, 2H), 2.20(m, 5H), 2.05 (m, 2H), 1.98 (m, 2H), 1.77 (m, 2H). LC-MS: m/z(M+H)=535.7

Compound 254

Step A: methyl 2-(4-bromopyridin-2-yl)acetate

To a mixture of 4-bromo-2-methylpyridine (1 g, 5.8 mmol) in 15 mL ofanhydrous THF was added LDA (9.2 mL, 2M) dropwise at −70, stirred for 30min dimethyl carbonate (630 mg, 7.0 mmol) was added dropwise to theabove solution. After stirring for another 1 h, LC-MS found the reactionfinished. It was quenched by Sat. NH4Cl solution and extracted withEtOAc. The organic layer was separated and evaporated under reducedpressure. The crude product was purified by a standard method to givedesired product. LC-MS: m/z (M+H) 231.1.

Step B: methyl 2-(4-(azetidin-1-yl)pyridin-2-yl)acetate

To a solution of (500 g, 2.2 mmol) in a mixture of dioxane (10 ml) andwere added Pd2(dba)3 (200 mg, 0.22 mmol) and Xantphos (185 mg, 0.22mmol), Cs2CO3 (1.4 g, 4.3 mmol) followed by azetidine (135 mg, 2.4 mmol)under nitrogen atmosphere, and the mixture was heated overnight at90.deg.C. After cooling to ambient temperature, the separated organiclayer was evaporated under reduced pressure. The residue was taken upinto ethyl acetate, washed with aqueous potassium carbonate solution andbrine, and dried over sodium sulfate. After evaporation, the residue waspurified by a standard method to give methyl2-(4-(azetidin-1-yl)pyridin-2-yl)acetate.

¹H NMR (CHLOROFORM-d) δ: 8.02 (d, J=7.0 Hz, 1H), 6.36 (d, J=2.4 Hz, 1H),6.27 (dd, J=6.7, 2.4 Hz, 1H), 4.81 (s, 2H), 4.25 (t, J=7.7 Hz, 4H), 3.38(s, 3H), 2.60 (m, 2H). LC-MS: m/z (M+H) 207.3

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(azetidin-1-yl)pyridin-2-yl)acetamide)(Compound 254)

The procedure was the same as Step B of Compound 249

¹H NMR (DMSO-d₆) δ: 8.06 (d, J=5.9 Hz, 2H), 6.41 (d, J=1.9 Hz, 2H), 6.30(dd, J=5.9, 2.1 Hz, 2H), 3.91-4.02 (t, 8H, J=7.2 Hz), 3.87 (s, 4H),3.48-3.52 (m, 2H), 2.36-2.41 (m, 4H), 2.31 (t, 2H, J=4 Hz), 1.94 (m,2H), 1.85 (m, 2H), 1.63 (m, 2H). LC-MS: m/z (M+H) 631.2

Compound 255

The procedure was the same as Compound 254

Step A: methyl 2-(4-(3-hydroxyazetidin-1-yl)pyridin-2-yl)acetate

¹H NMR (methanol-d4, 400 MHz) δ 8.03 (d, J=6.8 Hz, 1H), 6.54 (d, J=2.4Hz, 1H), 6.50 (dd, J=6.8 Hz, 2.4 Hz, 1H), 4.89 (s, 2H), 4.80-4.77 (m,1H), 4.22-4.38 (m, 2H), 3.96-3.92 (m, 2H), 3.75 (s, 3H). LC-MS: m/z(M+H) 223.4

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(3-hydroxyazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 255)

¹H NMR (methanol-d4, 400 MHz) δ 8.04 (d, J=6.80 Hz, 2H), 6.28 (d, J=2.0Hz, 2H), 6.18 (dd, J=6.0 Hz, 2.0 Hz, 2H), 4.72-4.69 (m, 2H), 4.20-4.17(m, 4H), 4.06 (s, 4H), 3.81-3.75 (m, 4H), 3.50-3.47 (m, 2H), 2.39 (t,J=5.6 Hz, 2H), 1.99-1.90 (m, 4H), 1.71 (t, J=5.6 Hz, 2H). LC-MS: m/z(M+H) 664.1

Compound 256

The procedure was the same as Compound 254

Step A: Methyl 2-(4-(3-fluoroazetidin-1-yl)pyridin-2-yl)acetate

¹H NMR (CDCl₃, 400 MHz) δ 8.21 (d, J=5.6 Hz, 1H), 6.33 (d, J=2.0 Hz,1H), 6.26 (dd, J=6.0 Hz, 2.0 Hz, 1H), 5.58-5.40 (m, 1H), 4.36-4.27 (m,2H), 4.19-4.09 (m, 2H), 3.83 (s, 2H), 3.75 (s, 3H). LC-MS: m/z (M+H)225.4.

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(3-fluoroazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 256)

¹H NMR (METHANOL-d₄) δ 8.07 (s, 2H), 6.32-6.23 (m, 4H), 5.37 (d, J=60Hz, 2H), 4.26 (m, 4H), 4.10 (m, 4H), 3.87 (s, 4H), 3.40 (m, 2H), 2.28(t, J=5.6 Hz, 2H), 1.87 (m, 4H), 1.60 (m, 2H). LC-MS: m/z (M+H) 668.0.

Compound 257

The procedure was the same as Compound 249

Step A: methyl 2-(3-(azetidin-1-yl)phenyl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.12-7.22 (s, 1H), 6.66 (d, J=7.5 Hz, 1H),6.31-6.42 (m, 2H), 3.90 (t, J=7.3 Hz, 4H), 3.71 (s, 3H), 3.58 (s, 2H),2.38 (quin, J=7.2 Hz, 2H). LC-MS: m/z (M+H)=206.5

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(azetidin-1-yl)phenyl)acetamide)(Compound 257)

¹H NMR (DMSO-d₆) δ: 12.66 (s, 2H), 7.10 (t, J=7.8 Hz, 2H), 6.62 (d,J=7.5 Hz, 2H), 6.38 (s, 2H), 6.30 (dd, J=7.9, 1.7 Hz, 2H), 3.78 (t,J=7.3 Hz, 8H), 3.68 (s, 4H), 3.44-3.49 (m, 2H), 2.25-2.34 (m, 6H),1.89-1.98 (m, 2H), 1.76-1.87 (m, 2H), 1.53-1.66 (m, 2H). LC-MS: m/z(M+H)=629.8

Compound 258

The procedure was the same as Compound 254

Step A: Methyl 2-(4-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetate

¹H NMR (METHANOL-d₄) δ 8.29 (d, J=6.4 Hz, 2H), 6.73 (d, J=2.4 Hz, 2H),6.66 (dd, J=6.4 Hz, 2.4 Hz, 2H), 4.82-4.79 (m, 2H), 4.72-4.70 (m, 2H),3.81 (s, 2H), 3.74 (s, 3H). LC-MS: m/z (M+H)=243.4.

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 258)

¹H NMR (METHANOL-d₄) δ 8.20 (d, J=6.4 Hz, 2H), 6.74 (d, J=2.4 Hz, 2H),6.68 (dd, J=6.4 Hz, 2.4 Hz, 2H), 4.62-4.56 (m, 8H), 3.57 (t, J=5.6 Hz,2H), 2.44 (t, J=5.6 Hz, 2H), 2.09-2.04 (m, 2H), 1.99-1.96 (m, 2H),1.76-1.74 (m, 2H). LC-MS: m/z (M+H)=703.2.

Compound 259

Step A: methyl 2-(3-(4-methylpiperazin-1-yl)phenyl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.21 (t, J=7.9 Hz, 1H), 6.81-6.86 (m, 2H), 6.77(d, J=7.3 Hz, 1H), 3.68 (s, 3H), 3.58 (s, 2H), 3.19-3.27 (m, 4H),2.55-2.61 (m, 4H), 2.37 (s, 3H). LC-MS: m/z (M+H)=249.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-4-methylpiperazin-1-yl)phenyl)acetamide)(Compound 259)

To a solution of5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (50 mg,0.19 mmol) in 3 mL of DMSO were added methyl2-(3-(4-methylpiperazin-1-yl)phenyl)acetate (140 mg, 0.57 mmol) andpotassium 2-methylpropan-2-olate (63 mg, 0.57 mmol) under nitrogenatmosphere, and the mixture was stirred for 40 min at 100.deg. C. undermicrowave irradiation. The reaction mixture was poured into water andextracted with ethyl acetate. The organic layer was washed withsaturated aqueous sodium bicarbonate and brine and dried over sodiumsulfate. The solvent was evaporated under reduced pressure. The residuewas purified by a standard method to give the desired compound.

¹H NMR (METHANOL-d₄) δ: 7.18-7.25 (m, 2H), 6.98 (br. s., 2H), 6.91 (d,J=8.3 Hz, 2H), 6.84 (d, J=7.0 Hz, 2H), 3.77 (d, J=2.4 Hz, 4H), 3.42-3.52(m, 2H), 3.18-3.26 (m, 8H), 2.61-2.69 (m, 8H), 2.37 (s, 6H), 2.30 (t,2H, J=4.0 Hz), 1.88-1.97 (m, 2H), 1.78-1.88 (m, 2H), 1.53 (m, 2H).LC-MS: m/z (M+H) 715.3

Compound 260

The procedure was the same as Compound 259

Step A: methyl 2-(3-(pyrrolidin-1-yl)phenyl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.20 (dd, J=8.7, 7.4 Hz, 1H), 6.61 (d, J=7.0Hz, 1H), 6.51 (br. s., 2H), 3.71 (s, 3H), 3.60 (s, 2H), 3.3 (m, 4H),1.98-2.06 (m, 4H). LC-MS: (M+H) m/z 220.3

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(pyrrolidin-1-yl)phenyl)acetamide)(Compound 260)

¹H NMR (DMSO-d₆) δ: 12.65 (s, 2H), 7.09 (t, J=7.8 Hz, 2H), 6.49-6.57 (m,4H), 6.42 (d, J=8.3 Hz, 2H), 3.68 (s, 4H), 3.47 (m, 2H), 3.15-3.23 (t,8H, J=8 Hz), 2.29 (t, 2H, J=4 Hz), 1.94 (m, 10H), 1.84 (m, 2H), 1.61 (m,2H). LC-MS: (M+H) m/z 657.3

Compound 261

The procedure was the same as Compound 259.

Step A: methyl 2-(3-(cyclopropylamino)phenyl)acetate

¹H NMR (CHLOROFORM-d): 7.17 (t, J=7.7 Hz, 1H), 6.74-6.79 (m, 2H), 6.69(d, J=7.5 Hz, 1H), 3.71 (s, 3H), 3.58 (s, 2H), 2.46 (dt, J=6.6, 3.3 Hz,1H), 0.72-0.78 (m, 2H), 0.51-0.59 (m, 2H). LC-MS: m/z (M+H) 206.3

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(cyclopropylamino)phenyl)acetamide)(Compound 261)

¹H NMR (DMSO-d₆) δ: 12.73 (br. s., 2H), 7.04 (t., J=4.0 Hz, 2H),6.61-6.53 (m, 6H), 6.10 (s, 2H), 3.65 (s, 4H), 3.48 (m, 2H), 2.29 (m,4H), 1.92 (m, 2H), 1.84 (m, 2H), 1.60 (m, 2H), 0.67 (m, 4H), 0.34 (m,4H). LC-MS: m/z (M+H) 628.3

Compound 262

The procedure was the same as Compound 259

Step A: methyl 2-(3-morpholinophenyl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.21 (t, J=7.9 Hz, 1H), 6.82-6.86 (m, 2H), 6.77(d, J=7.3 Hz, 1H), 3.68 (s, 3H), 3.74 (t, J=8 Hz, 4H), 3.58 (s, 2H),3.10 (t, J=8 Hz, 4H). LC-MS: m/z (M+H) 235.3

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-morpholinophenyl)acetamide)(Compound 262)

¹H NMR (DMSO-d₆) δ: 12.67 (br. s, 2H), 7.1.7 (t, J=7.9 Hz, 2H), 6.92 (s,2H), 6.84 (dd, J=8.2, 2.0 Hz, 2H), 6.76 (d, J=7.5 Hz, 2H), 3.67-3.80 (m,12H), 3.43-3.50 (m, 2H), 3.05-3.14 (m, 8H), 2.29 (t. 2H, J=4 Hz),1.87-1.98 (m, 2H), 1.83 (m, 2H), 1.61 (m, 2H). LC-MS: m/z 689.3 (M+H).

Compound 263

Step A to Step B: ditert-butyl4,4′-(3,3′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(3,1-phenylene))dipiperazine-1-carboxylate

The procedure was the same as Compound 259

¹H NMR (DMSO-d₆) δ: 12.67 (s, 2H), 7.17 (t, J=7.9 Hz, 2H), 6.94 (s, 2H),6.82-6.90 (m, 2H), 6.77 (d, J=7.5 Hz, 2H), 3.72 (s, 4H), 3.41-3.51 (m,10H), 3.00-3.14 (t, 8H, J=5.2 Hz), 2.30 (t, J=5.6 Hz, 2H), 1.89-1.99 (m,4H), 1.80-1.88 (m, 2H), 1.42 (s, 18H). LC-MS: m/z (M+H) 888.1

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(piperazin-1-yl)phenyl)acetamide)(Compound 263)

The procedure was the same as Step C of Compound 239.

¹H NMR (DMSO-d6) δ: 7.15 (t, J=7.8 Hz, 2H), 6.91 (s, 2H), 6.79-6.85 (m,2H), 6.73 (d, J=7.5 Hz, 2H), 4.15 (br, 2H), 3.68 (s, 4H), 3.47 (m, 2H),3.01-3.11 (t, 8H, J=4.8 Hz), 2.80-2.93 (t, 8H, J=4.8 Hz), 2.29 (t, 2H,J=9.6 Hz), 1.92 (m, 2H), 1.95 (m, 2H), 1.61 (m, 2H). LC-MS: 687.3 (M+H)m/z

Compound 264

The procedure is same to the reaction to make Compound 263.

Step B: di-tert-butyl((((5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl))bis(2-oxoethane-2,1-diyl))bis(3,1-phenylene))bis(methylcarbamate)

¹H NMR (DMSO-d₆) δ: 12.73 (br. s., 1H), 7.27-7.33 (m, 2H), 7.24 (s, 2H),7.18 (d, J=8.1 Hz, 2H), 7.13 (d, J=7.5 Hz, 2H), 3.79 (s, 4H), 3.47 (m,2H), 3.17 (s, 6H), 2.28 (t, 2H, J=8 Hz), 1.93-1.83 (m, 4H), 1.61 (m,2H), 1.36 (s, 18H). LC-MS: m/z 778.2 (M+H)

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(methylamino)phenyl)acetamide)(Compound 264)

¹H NMR (DMSO-d6) δ: 12.65 (s, 2H), 7.02 (t, J=7.9 Hz, 2H), 6.47-6.54 (m,4H), 6.42 (d, J=7.3 Hz, 2H), 4.16 (br, 2H), 3.64 (s, 4H), 3.46 (m, 2H),2.65 (s, 6H), 2.29 (t, 2H, J=4 Hz), 1.88-1.98 (m, 2H), 1.78-1.88 (m,2H), 1.61 (m, 2H). LC-MS: m/z (M+H) 577.9

Compound 265

The procedure was the same as Compound 249

Step A: ethyl 2-(4-morpholinophenyl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.193 (d, 2H); 6.872 (d, 2H); 4.272 (q, J=7.14Hz, 2H); 3.853 (m, 2H); 3.533 (s, 2H); 3.140 (m, 2H); 1.247 (t, J=7.14,3H). LC-MS m/z 250.1 (M+H).

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-morpholinophenyl)acetamide)(Compound 265)

¹H NMR (CHLOROFORM-d) δ: 7.45 (d, J=6.4 Hz, 4H), 7.20 (m, 4H), 4.03 (m,8H), 3.98 (m, 4H), 3.54-3.60 (m, 2H), 3.30 (m, 8H), 2.49 (t, J=5.4 Hz,2H), 2.05 (m, 2H), 1.96 (m, 2H), 1.76 (m, 2H). LC-MS: m/z (M+H)=689.9

Compound 266

The procedure was the same as Compound 249

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(4-(3,3-difluoroazetidin-1-yl)phenyl)acetamide)(Compound 266)

¹H NMR (DMSO-d₆) δ: 12.63 (s, 2H), 7.20 (d, J=8.3 Hz, 4H), 6.54 (d,J=8.3 Hz, 4H), 4.23 (t, J=12.2 Hz, 8H), 3.68 (s, 4H), 3.46 (m, 2H), 2.28(t, J=5.6 Hz, 2H), 1.92 (m, 2H), 1.83 (m, 2H), 1.60 (m, 2H). LC-MS: m/z(M+H)=701.9

Compound 267

The procedure was the same as Compound 249

Step A: methyl 2-(6-(azetidin-1-yl)pyridin-2-yl)acetate

LC-MS: m/z (M+H)=207.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(azetidin-1-yl)pyridin-2-yl)acetamide)(Compound 267)

¹H NMR (DMSO-d₆) δ: 12.70 (s, 2H), 7.47 (dd, J=8.2, 7.4 Hz, 2H), 6.61(d, J=7.3 Hz, 2H), 6.24 (d, J=8.1 Hz, 2H), 3.90 (t, J=7.4 Hz, 8H), 3.81(s, 4H), 3.44-3.55 (m, 2H), 2.24-2.37 (m, 6H), 1.90-2.02 (m, 2H),1.77-1.90 (m, 2H), 1.55-1.69 (m, 2H). LC-MS: m/z (M+H)=631.8.

Compound 268

The procedure was the same as Compound 249

Step A: methyl 2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.50 (t, J=7.8 Hz, 1H), 6.72 (d, J=7.3 Hz, 1H),6.30 (d, J=8.1 Hz, 1H), 4.35 (t, J=12.1 Hz, 4H), 3.73 (s, 2H), 3.75 (s,3H). LC-MS: m/z (M+H)=243.4

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 268)

¹H NMR (DMSO-d₆) δ: 12.70 (s, 2H), 7.59 (dd, J=7.3, 8.3 Hz, 2H), 6.79(d, J=7.3 Hz, 2H), 6.48 (d, J=8.3 Hz, 2H), 4.34 (t, J=12.5 Hz, 8H), 3.87(s, 4H), 3.45-3.52 (m, 2H), 2.32 (t, J=5.9 Hz, 2H), 1.95 (m, 2H),1.78-1.89 (m, 2H), 1.62 (m, 2H). LC-MS: m/z (M+H)=703.9

Compound 269

The procedure was the same as Compound 249

Step A: methyl 2-(3-(3,3-difluoroazetidin-1-yl)phenyl)acetate

¹H NMR (CHLOROFORM-d) δ: 7.19-7.28 (m, 1H), 6.80 (d, J=7.5 Hz, 1H), 6.48(m, 2H), 4.26 (t, J=11.8 Hz, 4H), 3.72 (s, 3H), 3.61 (s, 2H). LC-MS: m/z(M+H)=242.4

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(3,3-difluorocyclobutyl)phenyl)acetamide)(Compound 269)

¹H NMR (DMSO-d₆) δ: 12.68 (s, 2H), 7.18 (t, J=7.8 Hz, 2H), 6.76 (d,J=7.5 Hz, 2H), 6.54 (s, 2H), 6.48 (d, J=7.5 Hz, 2H), 4.25 (t, J=12.2 Hz,8H), 3.73 (s, 4H), 3.48 (m, 2H), 2.29 (t, J=5.4 Hz, 2H), 1.88-1.99 (m,2H), 1.77-1.87 (m, 2H), 1.61 (m, 2H). LC-MS: m/z (M+H)=701.9

Compound 270

The procedure was the same as Compound 249

Step A: methyl 2-(6-(3-methoxyazetidin-1-yl)pyridin-2-yl)acetate

LC-MS: m/z (M+H)=237.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3-methoxyazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 270)

¹H NMR (DMSO-d₆) δ: 12.69 (s, 2H), 7.49 (dd, J=7.3, 8.3 Hz 2H), 6.64 (d,J=7.3 Hz, 2H), 6.30 (d, J=8.3 Hz, 2H), 4.26-4.33 (m, 2H), 4.04-4.16 (m,4H), 3.81 (s, 4H), 3.71 (dd, J=8.9, 4.0 Hz, 4H), 3.44-3.54 (m, 2H), 3.34(s, 6H), 2.32 (d, J=5.4 Hz, 2H), 1.91-2.00 (m, 2H), 1.78-1.90 (m, 2H),1.63 (d, J=5.4 Hz, 2H). LC-MS: m/z (M+H)=691.8

Compound 271

Step A: methyl 2-(6-chloropyridin-3-yl)acetate

To a solution of 2-(6-chloropyridin-3-yl)acetic acid (2 g, 117 mmol) inmethanol (40 ml) was added SOCl₂ (1.38 g, 117 mmol). The reactionmixture was concentrated after two hours to give the desired product.LC-MS: m/z (M+H)=186.6

Step B: methyl 2-(6-(3,3-difluoroazetidin-1-yl)pyridin-3-yl)acetate

To a sealed microwave reaction vessel containing methyl2-(6-chloropyridin-3-yl)acetate (200 mg, 1.08 mmol),3,3-difluoroazetidine hydrochloride (154 mg, 1.19 mmol), Xantphos (93mg, 0.162 mmol), Cs2CO3 (1.054 g, 3.23 mmol), and Pd2(dba)3 (99.0 mg,0.11 mmol) was added 3.0 ml of dioxane. The reaction mixture wasmicrowave radiated at 90 C for 60.0 min. It was cooled to roomtemperature and diluted with CH₂Cl₂ (20.0 ml). The suspension wasfiltered, washed with CH₂Cl₂ (3×10.0 ml), and the filtrate was washedwith water and brine. The organic layer was dried over anhydrous Na2SO4,filtered. The filtrate was concentrated. The residue was purified by astandard method to give the title compound.

¹H NMR (CHLOROFORM-d) δ: 8.09 (d, J=1.9 Hz, 1H), 7.54 (dd, J=8.3, 2.1Hz, 1H), 6.42 (d, J=8.3 Hz, 1H), 4.41 (t, J=12.0 Hz, 4H), 3.72 (s, 3H),3.55 (s, 2H). LC-MS: m/z (M+H)=243.3

Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3,3-difluoroazetidin-1-yl)pyridin-3-yl)acetamide)(Compound 271)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 13.72 (br. s., 2H), 8.24 (d, J=1.9 Hz, 2H),7.65 (dd, J=8.6, 2.1 Hz, 2H), 6.34 (d, J=8.3 Hz, 2H), 4.30 (t, J=12.1Hz, 8H), 3.94 (s, 4H), 3.55-3.63 (m, 2H), 2.50 (t, J=5.4 Hz, 2H), 2.07(m, 2H), 1.96-2.01 (m, 2H), 1.73-1.82 (m, 2H). LC-MS: m/z (M+H)=703.8

Compound 272

Step A: tert-butyl4-(3-(2-methoxy-2-oxoethyl)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate

The solution of methyl 2-(3-bromophenyl)acetate (288.9 mg, 1.26 mmol),tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate(300 mg, 0.97 mmol), PdCl₂(dppf) (35.5 mg, 4.8% mmol) and K₂CO₃ (268.1mg, 1.94 mmol) in DMF (20 mL) was stirred at 90 degree under N₂overnight. After cooling to room temperature, the mixture was filteredwith celite, diluted with EtOAc, washed with water, dried over Na₂SO₄and evaporated under reduced pressure. The residue was purified by astandard method to get desired product. LC-MS: m/z (M+H)=332.4

Step B: tert-butyl4-(3-(2-methoxy-2-oxoethyl)phenyl)piperidine-1-carboxylate

The solution of tert-butyl4-(3-(2-methoxy-2-oxoethyl)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate(330 mg, 1.0 mmol) and Pd/C (30 mg) in methanol (10 mL) was stirredunder H₂ at room temperature for 12 h. Then, the reaction mixture wasfiltered and evaporated under reduced pressure to get desired productfor the next step without further purification. LC-MS: m/z (M+H)=334.4

Step C: tert-butyl4,4′-(3,3′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl)bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(3,1-phenylene))dipiperidine-1-carboxylate

The procedure was the same as Step B of Compound 84

LC-MS: m/z (M+H)=886.1

Step D:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(piperidin-4-yl)phenyl)acetamide)(Compound 272)

The solution of tert-butyl4,4′-(3,3′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(3,1-phenylene))dipiperidine-1-carboxylate(15 mg, 0.01 mmol) in HCl/MeOH (6 mL, 3 N) was stirred at roomtemperature for 6 h. The mixture was evaporated under reduced pressureto purify by a standard method.

¹H NMR (METHANOL-d₄) δ: 12.66 (s, 2H), 8.46 (br. s., 2H), 7.27 (t, J=7.8Hz, 2H), 7.19 (s, 2H), 7.13 (d, J=8.1 Hz, 2H), 3.72 (s, 4H), 3.41-3.45(m, 2H), 3.39 (d, J=12.6 Hz, 4H), 3.02 (td, J=13.0, 2.6 Hz, 4H),2.75-2.84 (m, 2H), 2.19-2.35 (t, J=5.4 Hz, 2H), 1.90-2.00 (m, 6H),1.73-1.85 (m, 6H), 1.54-1.67 (m, 2H). LC-MS: m/z (M+H)=686.0

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl)bis(2-(4-(piperidin-4-yl)phenyl)acetamide)(Compound 290)

The procedure was the same as Compound 272

¹H NMR (METHANOL-d₄) δ: 12.68 (s, 2H), 8.44 (br. s., 2H), 7.22 (d, J=8.6Hz, 4H), 7.15 (d, J=8.5 Hz, 4H), 3.70 (s, 4H), 3.41-3.46 (m, 2H), 3.39(d, J=12.6 Hz, 4H), 3.02 (td, J=13.0, 2.6 Hz, 4H), 2.75-2.84 (m, 2H),2.19-2.36 (t, J=5.4 Hz, 2H), 1.90-2.00 (m, 6H), 1.73-1.85 (m, 6H),1.55-1.68 (m, 2H). LC-MS: m/z (M+H)=686.0

Compound 273

Step A: methyl 2-(1H-imidazol-4-yl)acetate hydrochloride

To a solution of 2-(1H-imidazol-4-yl)acetic acid hydrochloride (200 mg,1.23 mmol) in methanol (5 ml) was added SOCl₂ (145 mg, 1.23 mmol). Thereaction mixture was concentrated after two hours to give the desiredproduct. LC-MS: m/z (M+H)=141.1

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1H-imidazol-4-yl)acetamide)(Compound 273)

A solution of5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (33 mg,0.119 mmol), methyl 2-(1H-imidazol-4-yl)acetate hydrochloride (50 mg,0.357 mmol), and cesium carbonate (300 mg, 1.19 mmol) inN,N-dimethylformamide (1 ml) was heated to 130° C. for 45 mins undermicrowave. Then the reaction mixture was cooled to room temperature andwas poured into to water. The mixture was extracted by ethyl acetate (50ml*3), the organic layer was washed by brine, dried by sodium sulfate,filtered, concentrated to give the residue; the residue was purified bya standard method to give the desired product.

¹H NMR (METHANOL-d₄) δ: 8.20 (br. s., 2H), 7.27 (br. s., 2H), 3.97 (s,4H), 3.56-3.60 (m, 2H), 2.45 (t, J=5.6 Hz, 2H), 2.05 (m, 2H), 2.00 (m,2H), 1.74-1.78 (m, 2H). LC-MS: m/z (M+H)=499.5

Compound 274

Step A: methyl 2-(2-aminothiazol-4-yl)acetate

The procedure is the same as Step A of Compound 273. LC-MS: m/z(M+H)=173.2.

Step B: methyl 2-(2-(tert-butoxycarbonylamino)thiazol-4-yl)acetate

A solution of di-tert-butyl dicarbonate (279 mg, 1.28 mmol) in toluene(3 ml) was added a vessel containing methyl2-(2-aminothiazol-4-yl)acetate (200 mg, 1.16 mmol), the reaction mixturewas heated at 85° C. for 24 h. LCMS showed that the desired product wasdetected, the mixture was concentrated to give the residue, the residuewas purified by a standard method to give the desired product. LC-MS:m/z (M+H)=273.3

Step C: 2-(2-(tert-butoxycarbonyl(methyl)amino)thiazol-4-yl)acetic acid

A solution of methyl2-(2-(tert-butoxycarbonyl(methyl)amino)thiazol-4-yl) acetate (360 mg,1.258 mmol), and LiOH.H₂O (106 mg, 2.52 mmol) in THF:H₂O=6 ml:3 ml wasstirred at rt overnight. Then the reaction mixture was concentrated togive the desired product. LC-MS: m/z (M+H)=259.3

Step D: tert-butyl4,4′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl)bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(thiazole-4,2-diyl)bis(methylcarbamate)

The procedure was the same as Compound 37

¹H NMR (CHLOROFORM-d) δ: 6.78 (s, 2H), 3.92 (s, 4H), 3.54 (m, 2H), 2.41(t, J=5.5 Hz, 2H), 2.01 (m, 2H), 1.93 (m, 2H), 1.73-1.75 (m, 2H), 1.55(s, 18H). LC-MS: m/z (M+H)=591.8. LC-MS: m/z (M+H)=791.0

Step E:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-aminothiazol-4-yl)acetamide)(Compound 274)

The procedure was the same as Step C of Compound 239

¹H NMR (METHANOL-d₄) δ: 6.42 (s, 2H), 3.73 (s, 4H), 3.54-3.60 (m, 2H),2.44 (t, J=5.6 Hz, 2H), 2.03-2.07 (m, 2H), 1.96-2.01 (m, 2H), 1.74-1.78(m, 2H). LC-MS: m/z (M+H)=563.8

Compound 275

Step A: methyl 2-(2-(methylamino)thiazol-4-yl)acetate

A mixture of methyl 4-chloro-3-oxobutanoate (1 g, 11.1 mmol) and1-methylthiourea (1.72 g, 11.43 mmol) in MeOH (5 mL) was heated to 80°C. for 3 hours. The mixture was cooled, ethanol evaporated, and dilutedwith saturated sodium bicarbonate, and extracted with ethyl acetate. Theorganic layers were dried over magnesium sulfate and the reactionmixture purified by a standard method to provide methyl2-(2-(methylamino)thiazol-4-yl)acetate. LC-MS: m/z (M+H)=187.2

Step B to Step D: tert-butyl4,4′-(2,2′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl))bis(thiazole-4,2-diyl)bis(methylcarbamate)

The procedure was the same as Step B to Step D of Compound 274

¹H NMR (CHLOROFORM-d) δ: 6.75 (s, 2H), 3.85 (s, 4H), 3.65 (s, 6H),3.53-3.55 (m, 2H), 2.45 (t, J=5.5 Hz, 2H), 2.01 (m, 2H), 1.97 (m, 2H),1.73 (m, 2H), 1.58 (s, 18H). LC-MS: m/z (M+H)=791.0

Step E:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-(methylamino)thiazol-4-yl)acetamide)(Compound 275)

The procedure was the same as Step E of Compound 274

¹H NMR (METHANOL-d₄) δ: 6.46 (s, 2H), 3.78 (s, 4H), 3.54-3.60 (m, 2H),2.94 (s, 6H), 2.44 (t, J=5.5 Hz, 2H), 2.05 (m, 2H), 1.99 (m, 2H),1.73-1.78 (m, 2H). LC-MS: m/z (M+H)=591.8

Compound 276

Step A: methyl 2-(2-bromothiazol-4-yl)acetate

Methyl 2-(2-aminothiazol-4-yl)acetate (5 g, 26.8 mmol) was added undernitrogen to a solution of copper(11) bromide (6.77 g, 30 mmol) andf-butyl nitrite (4.79 ml, 40 mmol) in acetonitrile (20 ml) at −20° C.The reaction mixture was slowly warmed to room temperature and stirredfor two hours. The solution was then diluted with diethyl ether andwashed with 25 ml of 10 percent hydrochloric acid solution; the aqueousphase was extracted with 20 ml of diethyl ether. The combined organicphases were dried and evaporated to dryness. The residue was purified bya standard method to yield the title compound. LC-MS: m/z (M+H)==235.9

Step B: methyl 2-(2-morpholinothiazol-4-yl)acetate

The procedure was the same as Step A of Compound 249

¹H NMR (CHLOROFORM-d) δ: 6.47 (s, 1H), 3.80-3.85 (m, 4H), 3.75 (s, 3H),3.67 (s, 2H). LC-MS: m/z (M+H)=243.3.

Step C to Step D:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-morpholinothiazol-4-yl)acetamide)

The procedure was the same as Step C to Step D of Compound 274

¹H NMR (CHLOROFORM-d) δ: 6.50 (s, 2H), 3.85-3.93 (m, 12H), 3.64 (m, 8H),3.53-3.59 (m, 2H), 2.47 (t, J=5.4 Hz, 2H), 1.97-2.06 (m, 4H), 1.73-1.80(m, 2H). LC-MS: m/z (M+H)=703.9

Compound 277

The procedure was the same as Compound 276

Step A: methyl 2-(2-(3,3-difluoroazetidin-1-yl)thiazol-4-yl)acetate

¹H NMR (CHLOROFORM-d) δ: 6.53 (s, 1H), 4.42 (t, J=11.8 Hz, 4H), 3.73 (s,3H), 3.65 (s, 2H). LC-MS: m/z (M+H)=249.3

Step B to Step C:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-(3,3-difluoroazetidin-1-yl)thiazol-4-yl)acetamide)(Compound 277)

¹H NMR (CHLOROFORM-d) δ: 6.57 (s, 2H), 4.50 (t, J=11.7 Hz, 8H), 3.84 (s,4H), 3.54-3.61 (m, 2H), 2.47 (t, J=5.6 Hz, 2H), 2.01-2.05 (m, 2H), 1.98(m, 2H), 1.74-1.80 (m, 2H). LC-MS: m/z (M+H)=715.5.

Compound 278

Step A: methyl 3-oxo-4-thiocyanatobutanoate

A solution of methyl 4-chloro-3-oxobutanoate (10 g, 66 mmol) in toluene(100 ml) was added a vessel containing KSCN (9.6 g, 99 mmol), thereaction mixture was stirred at rt for 24 h. LCMS showed that thedesired product was detected, the mixture was concentrated to give theresidue, the residue was purified by a standard method to give thedesired product. LC-MS: m/z (M+H)=174.3

Step B: methyl 2-(2-(dimethylamino)thiazol-4-yl)acetate

A solution of methyl 3-oxo-4-thiocyanatobutanoate (350 mg, 2.02 mmol) inTHF (4 ml) was added a vessel containing dimethylamine in THF (2M; 1.01ml, 2.02 mmol), the reaction mixture was stirred at rt for 3 h. LCMSshowed that the desired product was detected, the mixture wasconcentrated to give the residue, the residue was purified by a standardmethod to give the desired product.

¹H NMR (CHLOROFORM-d) δ: 6.36 (s, 1H), 3.74 (s, 3H), 3.68 (s, 2H), 3.13(s, 6H). LC-MS: m/z (M+H)=201.3

Step C to Step D:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-(dimethylamino)thiazol-4-yl)acetamide)(Compound 278)

The procedure was the same as Step C to Step D of Compound 268

¹H NMR (METHANOL-d₄) δ: 6.51 (s, 2H), 3.79 (s, 4H), 3.54-3.61 (m, 2H),3.11 (s, 12H), 2.44 (t, J=5.8 Hz, 2H), 2.04-2.07 (m, 2H), 2.00 (m, 2H),1.74-1.79 (m, 2H). LC-MS: m/z (M+H)=619.9

Compound 279

Step A: sodium 2-(2-methoxythiazol-4-yl)acetate

A solution of methyl 2-(2-bromothiazol-4-yl)acetate (80 mg, 0.34 mmol),and sodium methanolate (300 mg) in MeOH was stirred at 85° C. overnight.Then the reaction mixture was concentrated to give the desired product.¹H NMR (METHANOL-d4) δ: 4.04 (s, 3H), 3.47 (d, J=1.1 Hz, 2H), 3.37 (s,1H). LC-MS: m/z (M+H)=174.2

Step B:N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-methoxythiazol-4-yl)acetamide)(Compound 279)

A solution of5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (10 mg,0.04 mmol), sodium 2-(2-methoxythiazol-4-yl)acetate (28 mg, 0.14 mmol),HATU (83 mg, 0.22 mmol), and DIPEA (30 mg, 0.234 mmol) inN,N-dimethylformamide (2 ml) was heated to 50° C. overnight. The mixturewas poured into water (20 ml), the precipitate was filtered to give thedesired product.

¹H NMR (CHLOROFORM-d) δ: 6.95-7.19 (s, 2H), 6.59 (s, 2H), 4.16-4.20 (m,6H), 3.84 (s, 4H), 3.55-3.62 (m, 2H), 2.48 (t, J=5.4 Hz, 2H), 2.04 (d,J=5.1 Hz, 2H), 1.96-2.00 (m, 2H), 1.74-1.80 (m, 2H). LC-MS: m/z(M+H)=593.6

Compound 79

Step A:N-(5-((1S,3S)-3-(5-amino-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide

A mixture of methyl 2-(pyridin-2-yl)acetate (107.0 mg, 0.71 mmol),5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (200mg, 0.71 mmol), cesium carbonate (762.7 mg, 1.42 mmol) in DMF (6 mL) washeated to 130° C. under nitrogen atmosphere and microwave for 45 min.The mixture was evaporated in vacuum to dryness. The residue waspurified by a standard method to afford desired compound.

¹H NMR (DMSO-d6) δ: 12.72 (s, 1H), 8.52 (d, J=4.0 Hz, 1H), 7.85 (td,J=7.7, 1.7 Hz, 1H), 7.48 (d, J=7.8 Hz, 1H), 7.37 (ddd, J=6.9, 5.7, 1.1Hz, 1H), 7.06 (s, 2H), 4.01 (s, 2H), 3.64 (m, 2H), 2.22 (t, J=5.8 Hz,2H), 1.76-1.91 (m, 4H), 1.60 (m, 2H). LC-MS: m/z (M+H)=402.2

Step B:2-(2-methoxyphenyl)-N-(5-((1S,3S)-3-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)acetamide(Compound 79)

A solution ofN-(5-((1S,3S)-3-(5-amino-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide(50 mg, 0.12 mmol), 2-(2-methoxyphenyl)acetic acid (31.0 mg, 0.19 mmol),HATU (71.0 mg, 0.19 mmol), and N-ethyl-N-isopropylpropan-2-amine (29.0mg, 0.22 mmol) in N,N-dimethylformamide (5 ml) was stirred at 50 degreeovernight. The mixture was poured into water (10 ml), the precipitatewas filtered to give the crude product. The crude product was purifiedby a standard method to give the desired product.

¹H NMR (METHANOL-d₄) δ: 8.52 (d, J=4.0 Hz, 1H), 7.85 (td, J=7.7, 1.7 Hz,1H), 7.48 (d, J=7.8 Hz, 1H), 7.37 (ddd, J=6.9, 5.7, 1.1 Hz, 1H),7.27-7.33 (m, 1H), 7.25 (d, J=7.3 Hz, 1H), 6.86-7.05 (m, 2H), 4.62 (s,4H), 3.83 (s, 3H), 3.53-3.64 (m, 2H), 2.44 (t, J=5.8 Hz, 2H), 1.91-2.11(m, 4H), 1.72-1.81 (m, 2H). LC-MS: m/z (M+H)=550.7

2-(3-methoxyphenyl)-N-(5-((1S,3S)-3-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)acetamide(Compound 81)

The procedure was the same as Compound 279

¹H NMR (DMSO-d₆) δ: 12.74 (s, 1H), 12.70 (s, 1H), 8.50 (d, J=4.8 Hz,1H), 7.67-7.87 (m, 1H), 7.40 (d, J=8.1 Hz, 1H), 7.27-7.32 (m, 1H), 7.24(t, J=7.9 Hz, 1H), 6.87-6.93 (m, 2H), 6.84 (d, J=8.3 Hz, 1H), 4.01 (s,2H), 3.77 (s, 2H), 3.74 (s, 3H), 3.48 (m, 2H), 2.31 (t, J=5.6 Hz, 2H),1.93 (m, 2H), 1.85 (m, 2H), 1.61 (m, 2H). LC-MS: m/z (M+H)=550.7

Compound 280

The procedure was the same as Compound 79

Step B:2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)-N-(5-((1S,3S)-3-(5-(2-(5-(2-methoxyethoxy)pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)acetamide(Compound 280)

¹H NMR (METHANOL-d₄) δ: 8.23 (d, J=2.7 Hz, 1H), 7.54-7.70 (m, 1H),7.30-7.54 (m, 2H), 6.81 (d, J=7.3 Hz, 1H), 6.49 (d, J=8.3 Hz, 1H), 4.38(t, J=12.2 Hz, 4H), 4.20-4.25 (m, 2H), 3.94 (s, 2H), 3.90 (s, 2H),3.73-3.82 (m, 2H), 3.52-3.61 (m, 2H), 3.44 (s, 3H), 2.45 (t, J=5.8 Hz,2H), 2.03-2.13 (m, 2H), 1.91-2.03 (m, 2H), 1.70-1.82 (m, 2H). LC-MS: m/z(M+H)=687.0

Compound 281

Step A:N-(5-((1S,3S)-3-(5-amino-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)acetamide

A solution of5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (200mg, 0.71 mmol), acetic acid (42.5 mg, 0.71 mmol), HATU (260.0 mg, 0.71mmol), and N-ethyl-N-isopropylpropan-2-amine (110.0 mg, 0.85 mmol) inN,N-dimethylformamide (10 ml) was stirred at room temperature overnight.The mixture evaporated in vacuum to dryness. The residue was purified bya standard method to afford desired compound. LC-MS: m/z (M+H)=325.4

Step B:N-(5-((1S,3S)-3-(5-acetamido-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide(Compound 281)

The procedure was the same as Step B of Compound 84

¹H NMR (CHLOROFORM-d) δ: 12.61 (br. s., 1H), 7.59 (t, J=7.8 Hz, 1H),6.73 (d, J=7.3 Hz, 1H), 6.39 (d, J=8.3 Hz, 1H), 4.59 (t, J=11.6 Hz, 4H),3.98 (s, 2H), 3.60 (m, 2H), 2.47 (s, 3H), 2.17-2.36 (m, 2H), 1.96 (m,2H), 1.66 (m, 2H), 1.34 (m, 2H. LC-MS: m/z (M+H)=535.7

Compound 282

The procedure was the same as Compound 281

Step A:N-(5-((1S,3S)-3-(5-amino-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)propionamide

LC-MS: m/z (M+H)=339.5

Step B:N-(5-((1S,3S)-3-(5-(2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)propionamide(Compound 282)

¹H NMR (DMSO-d₆) δ: 12.69 (s, 1H), 12.40 (s, 1H), 7.59 (dd, J=7.3, 8.1Hz, 1H), 6.80 (d, J=7.3 Hz, 1H), 6.48 (d, J=8.1 Hz, 1H), 4.34 (t, J=12.5Hz, 4H), 3.86 (s, 2H), 3.49 (m, 2H), 2.44-2.49 (q, J=7.5 Hz, 2H), 2.31(t, J=5.6 Hz, 2H), 1.90-2.01 (m, 2H), 1.86 (m, 2H), 1.57-1.66 (m, 2H),1.05-1.14 (t, 3H). LC-MS: m/z (M+H)=549.7

Compound 283

The procedure was the same as Compound 281

Step A:N-(5-((1S,3S)-3-(5-amino-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)cyclopropanecarboxamide

LC-MS: m/z (M+H)=351.4

Step B:N-(5-((1S,3S)-3-(5-acetamido-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide(Compound 283)

¹H NMR (CHLOROFORM-d) δ: 7.58 (t, J=7.8 Hz, 1H), 6.73 (d, J=7.3 Hz, 1H),6.38 (d, J=8.3 Hz, 1H), 4.57 (t, J=11.7 Hz, 4H), 3.99 (br. s., 2H),3.49-3.69 (m, 2H), 2.38-2.55 (m, 2H), 2.29-2.38 (m, 1H), 1.90-2.12 (m,4H), 1.64-1.84 (m, 2H), 1.17-1.23 (m, 2H), 1.05-1.13 (m, 2H). LC-MS: m/z(M+H)=561.6

N-(5-((1S,3S)-3-(5-acetamido-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(3-methoxyphenyl)acetamide(Compound 90)

A solution of5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine) (50 mg,0.18 mmol), acetic acid (1 eq), HATU (202 mg, 0.53 mmol), and DIPEA (73mg, 0.57 mmol) in N,N-dimethylformamide (2 ml) was heated to 50° C. for2 h, then 3-methoxy-2-phenylacetic acid was added. The mixture washeated at 50° C. overnight and then poured into water (20 ml), theprecipitate was filtered to give the crude product. The crude productwas purified by a standard method to give the desired product.

¹H NMR (CHLOROFORM-d) δ: 7.22 (s, 1H), 7.00-7.06 (m, 2H), 6.78-6.82 (m,1H), 4.01 (s, 2H), 3.76 (s, 3H), 3.60 (m, 2H), 2.47-2.50 (m, 5H),2.02-2.09 (m, 2H), 1.99 (m, 2H), 1.79 (m, 2H). LC-MS: m/z (M+H)=473.6

N-(5-((1S,3S)-3-(5-acetamido-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(2-methoxyphenyl)acetamide(Compound 94)

The procedure was the same as Compound 90

¹H NMR (CHLOROFORM-d) δ: 7.29-7.37 (m, 2H), 6.95-7.03 (m, 2H), 3.93 (s,3H), 3.90 (s, 2H), 3.53-3.61 (m, 2H), 2.46 (m, 5H), 1.95-2.06 (m, 4H),1.74-1.79 (m, 2H). LC-MS: m/z (M+H)=473.6

N-(5-((1S,3S)-3-(5-acetamido-1,3,4-thiadiazol-2-yl)cyclohexyl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide(Compound 93)

The procedure was the same as Compound 90

¹H NMR (CHLOROFORM-d) δ: 8.67 (d, J=4.3 Hz, 1H), 7.71-7.78 (m, 1H), 7.35(d, J=7.8 Hz, 1H), 7.31 (m, 1H), 4.10 (s, 2H), 3.55-3.64 (m, 2H), 2.47(m, 5H), 1.95-2.08 (m, 4H), 1.78 (m, 2H). LC-MS: m/z (M+H)=444.6

5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine)

Step A: (S,S)-dibenzyl cyclopentyl 1,3-dicarboxylate

To a mixture of cis and trans-cyclopentyl 1,3-dicarboxylic acid (1.58 g,10.0 mmol), Cs₂CO₃ (8.28 g, 25.5 mmol) in DMF (20 mL) was added BnBr(4.36 g, 25.5 mmol). The mixture was stirred at rt under nitrogen for 3h. The residue was diluted with water and extracted with ethyl acetate.The combined organic solution was washed with water, dried over Na₂SO₄and concentrated in vacuo. The crude product was purified by a standardmethod to afford trans-dibenzyl cyclopentyl 1,3-dicarboxylate.

Chiral SFC separation: cis- and trans-dibenzyl cyclopentyl1,3-dicarboxylate was separated by chiral SFC to afford (S,S)-dibenzylcyclohexyl 1,3-dicarboxylate.

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.43-7.34 (m, 10H), 5.15 (s, 4H),2.99-3.06 (m, 2H), 2.22 (t, J=7.8 Hz, 2H), 2.11 (m, 2H), 1.90 (m, 2H).LC-MS: m/z 171.2 (M−H)⁻. LC-MS: m/z (M+H)=423.6

Step B: (S,S)-cyclopentyl 1,3-dicarboxylic acid

To a solution of 0.5 g (S,S)-dibenzyl cyclopentyl 1,3-dicarboxylate in10 mL MeOH was added 10% Pd on carbon (0.05 g). The suspension wasflushed with hydrogen and stirred for 20 min. It was then filtered andconcentrated to give the desired compound. The configuration wasconfirmed by comparing the optical rotation of the product with standardvalue.

¹H NMR (DMSO-d₆) δ: 12.12 (br. s., 2H), 2.71-2.82 (m, 2H), 1.97 (t,J=7.8 Hz, 2H), 1.87-1.94 (m, 2H), 1.64-1.76 (m, 2H). LC-MS: m/z 157.2(M−H)⁻. [a]_(D) ²⁰=+32.8, c=5.0, H₂O [reported: [a]_(D) ²⁰=+32.5,reported in Aust. J. Chem.; 1979, 32, 2517].

Step C:5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine)

The compound was synthesized with a method similar to5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazol-2-amine).

¹H NMR (DMSO-d₆) δ: 7.06 (s, 4H), 3.49-3.55 (m, 2H), 2.22 (t, J=7.7 Hz,2H), 2.14-2.20 (m, 2H), 1.78-1.89 (m, 2H). LC-MS: m/z 269.3 (M+H)

N,N′-(5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(2-methoxyphenyl)acetamide)(Compound 284)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 7.14-7.32 (m, 4H), 6.85-7.03 (m, 4H), 3.54-3.83 (m,12H), 2.39 (t, J=7.7 Hz, 2H), 2.32 (m, 2H), 1.99 (m, 2H). LC-MS: m/z(M+H)=565.5

N,N′-(5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(3-(dimethylamino)phenyl)acetamide)(Compound 285)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆): 12.66 (br. s., 2H), 7.11 (t, J=7.8 Hz, 2H), 6.70 (s,2H), 6.61 (t, J=6.0 Hz, 4H), 3.72-3.82 (m, 6H), 2.88 (s, 12H), 2.38 (t,J=7.7 Hz, 2H), 2.29 (m, 2H), 1.93 (m, 2H). LC-MS: m/z (M+H)=591.5

N,N′-(5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-methoxypyridin-3-yl)acetamide)(Compound 286)

The procedure was the same as Compound 37

¹H NMR (DMSO-d₆) δ: 12.71 (s, 2H), 8.09 (d, J=1.9 Hz, 2H), 7.65 (dd,J=8.6, 2.4 Hz, 2H), 6.80 (d, J=8.6 Hz, 2H), 3.83 (s, 6H), 3.77 (s, 4H),3.74 (m, 2H), 2.38 (t, J=7.8 Hz, 2H), 2.24-2.34 (m, 2H), 1.89-2.02 (m,2H). LC-MS: m/z (M+H)=567.8

N,N′-(5,5′-((1S,3S)-cyclohexane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(5-methoxypyridin-2-yl)acetamide)(Compound 287)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.61 (s, 2H), 8.27 (d, J=5.6 Hz, 2H), 6.96 (d,J=2.4 Hz, 2H), 6.84 (dd, J=5.6, 2.4 Hz, 2H), 3.81 (s, 6H), 3.78 (s, 4H),3.61-3.69 (m, 2H), 2.31 (t, J=7.7 Hz, 2H), 2.21-2.28 (m, 2H), 1.88-1.96(m, 2H). LC-MS: m/z (M+H)=567.9

N,N′-(5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(3,3-difluoroazetidin-1-yl)pyridin-2-yl)acetamide)(Compound 288)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.67 (s, 2H), 7.59 (t, J=7.8 Hz, 2H), 6.79 (d,J=7.3 Hz, 2H), 6.48 (d, J=8.1 Hz, 2H), 4.34 (t, J=12.5 Hz, 8H), 3.86 (s,4H), 3.74-3.79 (m, 2H), 2.40 (t, J=7.7 Hz, 2H), 2.27-2.36 (m, 2H),1.89-2.02 (m, 2H). LC-MS: m/z (M+H)=689.8

N,N′-(5,5′-((1S,3S)-cyclopentane-1,3-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(6-(dimethylamino)pyridin-2-yl)acetamide)(Compound 289)

The procedure was the same as Step B of Compound 84

¹H NMR (DMSO-d₆) δ: 12.67 (s, 2H), 7.46 (t, J=7.9 Hz, 2H), 6.52 (d,J=8.6 Hz, 2H), 6.55 (d, J=7.3 Hz, 2H), 3.81 (s, 4H), 3.49 (m, 2H), 2.98(s, 12H), 2.38 (t, J=7.7 Hz, 2H), 2.22-2.33 (m, 2H), 1.85-2.01 (m, 2H).LC-MS: m/z (M+H)=593.9

Having thus described several aspects of several embodiments, it is tobe appreciated various alterations, modifications, and improvements willreadily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to be part of thisdisclosure, and are intended to be within the spirit and scope of theinvention. Accordingly, the foregoing description and drawings are byway of example only.

What is claimed is:
 1. A compound of formula (I) or a pharmaceuticallyacceptable salt thereof:

wherein X is C₃-C₇ cycloalkylene; each W, Y and Z is independently —S—,—CH═, —O—, —N═, or —NH—, provided that at least one of W, Y and Z is not—CH═; each R¹ and R² is independently —NH₂, —N(R³)—C(O)—R⁴,—C(O)—N(R³)—R⁴, —N(R³)—C(O)—O—R⁴, —N(R³)—C(O)—N(R³)—R⁴ or—N(R³)—C(O)—SR⁴; each R³ is independently hydrogen, C₁₋₆ alkyl or aryl;each R⁴ is independently C₁₋₆ alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl, cycloalkylalkyl, heterocyclylalkyl, orheterocyclyl, each of which is substituted with 0-3 occurrences of R⁵;each R⁵ is independently C₁₋₆ alkyl, C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆alkoxy, C₁₋₆ thioalkoxy, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇cycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclylalkyl, heterocyclyl, cyano, halo, oxo, —OH, —OCF₃, —OCHF₂,—SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl, —C(O)N(R⁷)₂,—N(R⁷)S(O)₁₋₂—C₁₋₆ alkyl, —S(O)₂N(R⁷)₂, —N(R⁷)₂, —C₁₋₆ alkylene-N(R⁷)₂,wherein said alkyl, C₁₋₆ alkoxy, —O—C₁₋₆ alkyleneC₁₋₆ alkoxy, C₁₋₆thioalkoxy, C₁₋₆ haloalkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkylalkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl,—SO₂—C₁₋₆ alkyl, —NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl, —C(O)N(R⁷)₂,—N(R⁷)S(O)₁₋₂—C₁₋₆ alkyl, —S(O)₂N(R⁷)₂, —N(R⁷)₂, or —C₁₋₆alkylene-N(R⁷)₂ is optionally substituted with 0-3 occurrences of R⁸; ortwo adjacent R⁵ moieties, taken together with the atoms to which theyare attached form a cycloalkyl or heterocyclyl; each R⁶ is independentlyhydrogen, fluoro, C₁₋₆ alkyl, —OH, —NH₂, —NH(CH₃), —N(CH₃)₂, or C₁₋₆alkoxy; each R⁷ is independently hydrogen or C₁₋₆ alkyl; each R⁸ isindependently halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OH, —N(R⁷)₂, or C₁₋₆alkoxy, —O—C₁₋₆ alkyleneC₁₋₆ alkoxy, CN, NO₂, —N(R⁷)—C(O)—C₁₋₆ alkyl,—C(O)N(R⁷)₂, —N(R⁷)S(O)₁₋₂C₁₋₆ alkyl, or —S(O)₂N(R⁷)₂; m is 0, 1, or 2;n is 0, 1, or 2; o is 1, 2 or 3; and p is 1, 2 or 3; provided that (1)when X is unsubstituted cyclopropyl, R¹ and R² are not both NH-phenyl;and (2) X is other than substituted cyclobutyl or substitutedcyclopentyl.
 2. The compound of claim 1, wherein each W is —S—, each Yis —N═ and each Z is —N═.
 3. The compound of claim 1, wherein o is 1 andp is
 1. 4. The compound of claim 1, wherein m is 0 and n is
 0. 5. Thecompound of claim 1, wherein n is 1 and m is
 1. 6. The compound of claim5, wherein each R⁶ is hydrogen.
 7. The compound of claim 1, wherein R¹and R² are the same.
 8. The compound of claim 1, wherein R¹ and R² aredifferent.
 9. The compound of claim 1, wherein R¹ and R² are each—N(R³)—C(O)—R⁴ wherein each R³ is hydrogen and each R⁴ is aralkyl orheteroaralkyl, each of which is substituted with 0-3 occurrences of R⁵.10. The compound of claim 1, wherein the compound is a compound ofFormula (II):


11. The compound of claim 10, wherein the compound is a compound ofFormula (IIa):


12. The compound of claim 1, wherein the compound is a compound ofFormula (III):


13. The compound of claim 1, wherein the compound is a compound ofFormula (IV) and q is 0, 1, 2, 3, or 4:


14. The compound of claim 1, wherein the compound is a compound ofFormula (IVa) and q is 0, 1, 2, 3, or 4:


15. The compound of claim 1, wherein the compound is a compound ofFormula (IVb) and q is 0, 1, 2, 3, or 4:


16. The compound of claim 1, wherein the compound is a compound ofFormula (IVc) and q is 0, 1, 2, 3, or 4:


17. A pharmaceutical composition comprising a compound of Formula (I) ora pharmaceutically acceptable salt thereof.
 18. A method of treatingcancer, the method comprising administering a compound of claim 1 or acomposition of claim 17 to a subject in need thereof.
 19. The method ofclaim 18, wherein the cancer is selected from a cancer characterized byi) a low level of E-cadherin expression compared to a referencestandard, ii) a high level of vimentin expression compared to areference standard, or iii) a low or decreased level of pyruvatecarboxylase expression.